Pde5 compositions and methods for immunotherapy

ABSTRACT

The present invention relates to compositions and methods for the regulated and controlled expression of proteins. Methods for inducing anti-cancer immune responses in a subject are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/621,593 filed Dec. 11, 2019, which is a 371 national phase of PCT/US2018/037005 filed Jun. 12, 2018, which claims priority to U.S. Provisional Application No. 62/518,078 filed on Jun. 12, 2017, U.S. Provisional Application No. 62/523,850 filed on Jun. 23, 2017, U.S. Provisional Application No. 62/523,862 filed on Jun. 23, 2017, and U.S. Provisional Application No. 62/555,313 filed on Sep. 7, 2017, the contents of each of which are herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2095_1003PCT_SL.txt, created on Jun. 12, 2018, which is 18,074,972 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to tunable biocircuit systems for the development of controlled and/or regulated therapeutic systems. In particular, regulatable biocircuits containing destabilizing domains (DD) derived from mutant human cGMP-specific phosphodiesterase type 5 (PDE5) are disclosed. The present invention also relates to compositions and methods for immunotherapy.

Provided in the present invention include polypeptides of biocircuit systems, effector modules, stimulus response elements (SREs) and immunotherapeutic agents, polynucleotides encoding the same, vectors and cells containing the polypeptides and/or polynucleotides for use in cancer immunotherapy. In one embodiment, the compositions comprise destabilizing domains (DDs) which tune protein stability.

BACKGROUND OF THE INVENTION

Safe and effective gene therapy requires tightly regulated expression of a therapeutic transgenic product (e.g., the protein product). Similarly, the analysis of gene function in development, cell differentiation and other physiological activities requires the controllable expression of a protein under investigation. However, current technologies do not allow titration of levels of target protein induced or kinetics of induction. Inadequate exogenous and/or endogenous gene control is a critical issue in numerous settings including ex vivo and in vivo gene therapy. This lack of tunability also makes it difficult to safely express proteins with narrow or uncertain therapeutic windows or those requiring more titrated, controlled or transient expression.

One approach to regulated protein expression or function is the use of Destabilizing Domains (DDs). Destabilizing domains are small protein domains that can be appended to a payload such as a protein of interest (POI). DDs render the attached protein of interest (POI) unstable in the absence of a DD-binding ligand such that the protein is rapidly degraded by the ubiquitin-proteasome system of the cell (Stankunas, K., et al., (2003). Mol. Cell 12, 1615-1624; Banaszynski, L. A., et al., (2006) Cell; 126(5): 995-1004; reviewed in Banaszynski, L. A., and Wandless, T. J. (2006) Chem. Biol. 13, 11-21; Iwamoto, M., et al. (2010). Chem Biol. 17(9):981-8; Egeler, E. L. et al. (2011). J Biol Chem. 286(36):31328-36; and Rakhit R, Navarro R, Wandless T J (2014) Chem Biol. September 18; 21(9):1238-52; Navarro, R. et al. (2016) ACS Chem Biol. 11(8): 2101-2104). In some cases, the protein of interest is not completely processed and may not be secreted or presented on the membrane in the absence of DD-binding ligand (Sellmeyer et al., (2012), doi.org/10.1371/journal.pone.0043297; the contents of which are incorporated by reference in their entirety). However, when a specific small molecule ligand binds its intended DD as a ligand binding partner, the instability is reversed and protein function is restored or, in some cases, processing is restored and the protein of interest is presented on the membrane or secreted. Such a system is herein referred to as a biocircuit, with the canonical DD-containing biocircuit described above being the prototypical model biocircuit

It is believed that improvements of biocircuits, including those containing DDs can form the basis of a new class of cell and gene therapies that employ tunable and temporal control of gene expression and function. Such novel moieties are described by the present inventors as stimulus response elements (SREs) which act in the context of an effector module to complete a biocircuit arising from a stimulus and ultimately producing a signal or outcome. When properly formatted with a polypeptide payload, and when activated by a particular stimulus, e.g., a small molecule, biocircuit systems can be used to regulate transgene and/or protein levels either up or down by perpetuating a stabilizing signal or destabilizing signal. This approach has many advantages over existing methods of regulating protein function and/or expression, which are currently focused on top level transcriptional regulation via inducible promoters.

The present invention provides novel protein domains, in particular, destabilizing domains (DDs) derived from mutant human cGMP-specific phosphodiesterase type 5 (PDE5), particularly the catalytic domain of human PDE5, that display small molecule dependent stability, and the biocircuit systems and effector modules comprising such DDs. Methods for tuning transgene functions using the same are also provided.

Cancer immunotherapy aims to eradicate cancer cells by rejuvenating the tumoricidal functions of tumor-reactive immune cells, predominantly T cells. Strategies of cancer immunotherapy including the recent development of checkpoint blockade, adoptive cell transfer (ACT) and cancer vaccines which can increase the anti-tumor immune effector cells have produced remarkable results in several tumors.

The impact of host anti-tumor immunity and cancer immunotherapy is impeded by three major hurdles: 1) low number of tumor antigen-specific T cells due to clonal deletion; 2) poor activation of innate immune cells and accumulation of tolerogenic antigen-presenting cells in the tumor microenvironment; and 3) formation of an immunosuppressive tumor microenvironment. Particularly, in solid tumors the therapeutic efficacy of immunotherapeutic regimens remains unsatisfactory due to lack of an effective an anti-tumor response in the immunosuppressive tumor microenvironment. Tumor cells often induce immune tolerance or suppression and such tolerance is acquired because even truly foreign tumor antigens will become tolerated. Such tolerance is also active and dominant because cancer vaccines and adoptive transfer of pre-activated immune effector cells (e.g., T cells), are subject to suppression by inhibitory factors in the tumor microenvironment (TME).

In addition, administration of engineered T cells could result in on/off target toxicities as well as a cytokine release syndrome (reviewed by Tey Clin. Transl. Immunol., 2014, 3: e17 10.1038).

Development of a tunable switch that can turn on or off the transgenic immunotherapeutic agent expression is needed in case of adverse events. For example, adoptive cell therapies may have a very long and an indefinite half-life. Since toxicity can be progressive, a safety switch is desired to eliminate the infused cells. Systems and methods that can tune the transgenic protein level and expression window with high flexibility can enhance therapeutic benefit, and reduce potential side effects.

In an effort to develop regulatable therapeutic agents for disease therapy, in particular cancer immunotherapy, the present invention provides biocircuit systems to control the expression of immunotherapeutic agents. The biocircuit system comprises a stimulus and at least one effector module that responds to the stimulus. The effector module may include a stimulus response element (SRE) that binds and is responsive to a stimulus and an immunotherapeutic agent operably linked to the SRE. In one example, a SRE is a destabilizing domain (DD) which is destabilized in the absence of its specific ligand and can be stabilized by binding to its specific ligand.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for immunotherapy. The compositions relate to tunable systems and agents that induce anti-cancer immune responses in a cell or in a subject. The tunable system and agent may be a biocircuit system comprising at least one effector module that is responsive to at least one stimulus. The biocircuit system may be, but is not limited to, a destabilizing domain (DD) biocircuit system, a dimerization biocircuit system, a receptor biocircuit system, and a cell biocircuit system. These systems are further taught in co-owned U.S. Provisional Patent Application No. 62/320,864 filed Apr. 11, 2016, 62/466,596 filed Mar. 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety).

The present invention provides compositions for an inducing immune response in a cell or subject. In some embodiments, the composition may include a stimulus response element (SRE) and at least one payload. In some embodiments, the payload may be attached, appended or associated with the SRE. The SRE may comprise a destabilizing domain (DD). In some embodiments, the DD may comprise, in whole or in part, the cGMP-specific 3′, 5′-cyclic phosphodiesterase (hPDE5; SEQ ID NO. 1). In some embodiments, the payload may be appended to the SRE.

In some embodiments, the DD may comprise the catalytic domain of hPDE5 (SEQ ID NO. 3). In some aspects, the catalytic domain may include amino acids 535-860 of hPDE5 (SEQ ID NO. 1). In some embodiments, the DD may include a mutation in the amino acid at position 732 (R732) of SEQ ID NO. 1. The mutation at position R732 may include but is not limited to R732L, R732A, R732G, R732V, R732I, R732P, R732F, R732W, R732Y, R732H, R732S, R732T, R732D, R732E, R732Q, R732N, R732M, R732C, and R732K.

In some aspects, the DD may be selected from the group including but not limited to hPDE5 (Amino acid 535-860 of WT, R732L) (SEQ ID NO. 12); hPDE5 (Amino acid 535-860 of WT, R732A) (SEQ ID NO. 384); hPDE5 (Amino acid 535-860 of WT, R732G) (SEQ ID NO. 383); hPDE5 (Amino acid 535-860 of WT, R732V) (SEQ ID NO. 385); hPDE5 (Amino acid 535-860 of WT, R732I) (SEQ ID NO. 386); hPDE5 (Amino acid 535-860 of WT, R732P) (SEQ ID NO. 387); hPDE5 (Amino acid 535-860 of WT, R732F) (SEQ ID NO. 388); hPDE5 (Amino acid 535-860 of WT, R732W) (SEQ ID NO. 389); hPDE5 (Amino acid 535-860 of WT, R732Y) (SEQ ID NO. 390); hPDE5 (Amino acid 535-860 of WT, R732H) (SEQ ID NO. 391); hPDE5 (Amino acid 535-860 of WT, R732S) (SEQ ID NO. 392); hPDE5 (Amino acid 535-860 of WT, R732T) (SEQ ID NO. 393); hPDE5 (Amino acid 535-860 of WT, R732D) (SEQ ID NO. 394); hPDE5 (Amino acid 535-860 of WT, R732E) (SEQ ID NO. 395); hPDE5 (Amino acid 535-860 of WT, R732Q) (SEQ ID NO. 396); hPDE5 (Amino acid 535-860 of WT, R732N) (SEQ ID NO. 397); hPDE5 (Amino acid 535-860 of WT, R732M) (SEQ ID NO. 398); hPDE5 (Amino acid 535-860 of WT, R732C) (SEQ ID NO. 399); and hPDE5 (Amino acid 535-860 of WT, R732K) (SEQ ID NO. 400).

In some embodiments, the mutation in the amino acid at position R732 may be R732L. In one embodiment, the DD may comprise the amino acid sequence of SEQ ID NO. 12. In some embodiments, the mutation in the amino acid at position R732 may be R732A. In one embodiment, the DD may comprise the amino acid sequence of SEQ ID NO. 384. In some embodiments, the mutation in the amino acid at position R732 may be R732G. In one embodiment, the DD may comprise the amino acid sequence of SEQ ID NO. 383.

The DDs of the present invention may further comprise one or more mutations independently selected from the group consisting of H653A, F736A, D764A, D764N, Y612F, Y612W, Y612A, W853F, I821A, Y829A, F787A, D656L, Y728L, M625I, E535D, E536G, Q541R, K555R, F559L, F561L, F564L, F564S, K591E, N587S, K604E, K608E, N609H, K630R, K633E, N636S, N661S, Y676D, Y676N, C677R, H678R, D687A, T712S, D724N, D724G, L738H, N742S, A762S, D764G, D764V, S766F, K795E, L797F, I799T, T802P, S815C, M816A, I824T, C839S, K852E, S560G, V585A, I599V, I648V, S663P, L675P, T711A, F744L, L746S, F755L, L804P, M816T, and F840S.

In one embodiment the DD may include the mutation H653A. In one embodiment, the DD may comprise the amino acid sequence of SEQ ID NO. 509. In one embodiment, the DD may include the mutation F736A. In one embodiment, the DD may comprise the amino acid sequence of SEQ ID NO. 227. In one embodiment, the DD may comprise the mutation D764A. In one embodiment, the DD comprises the amino acid sequence of SEQ ID NO. 510. In one embodiment, the DD may include the mutation Y612F. In one aspect, the DD may comprise the amino acid sequence of SEQ ID NO. 506. In one embodiment, the DD may include the Y612W mutation. In one aspect, the DD may include the amino acid sequence of SEQ ID NO. 507. In one embodiment, the DD may include the mutation Y612A. In one embodiment the DD may comprise the amino acid sequence of SEQ ID NO. 508. In one embodiment, the DD may include the mutation D64N. In some aspects, the DD may comprise the amino acid sequence of SEQ ID NO. 505.

In some embodiments, the SRE of the present invention may be appended to a payload. In some aspects, the payload may be an immunotherapeutic agent. In some aspects, the immunotherapeutic agent may be selected from a chimeric antigen receptor (CAR) and a cytokine-cytokine receptor fusion polypeptide.

In some embodiments the CAR may include an extracellular target moiety, a hinge and transmembrane domain, an intracellular signaling domain; and optionally, one or more co-stimulatory domains. In some embodiments, the CAR may be a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR.

The extracellular target moiety of the CAR may have an affinity of bind to a target molecule on the surface of the cancer cell. In some aspects, the extracellular target moiety of the CAR may be an scFv. In one aspect, the target molecule may be CD19. In some embodiments, the extracellular target moiety of the CAR is a CD19 scFv (SEQ ID NO. 8233). In some embodiments, wherein the hinge and transmembrane domain of the CAR may be paired. The paired hinge and transmembrane domain may be derived from CD8a, CDS, CD4, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD148, DAP 10, EpoRI, GITR, LAG3, ICOS, Her2, OX40 (CD134), 4-1BB (CD137), CD152, CD154, PD-1, or CTLA-4. In some embodiments, the paired domain may be derived from a transmembrane region of an alpha, beta or zeta chain of a T-cell receptor; or an immunoglobulin selected from IgG1, IgD, IgG4, and an IgG4 Fc region; or the CD3 epsilon chain of a T-cell receptor. In one embodiment, the paired hinge and transmembrane domain of the CAR may be derived from CD8a. In one aspect, the paired hinge and transmembrane domain may comprise the amino acid sequence of SEQ ID NO. 8235. In some embodiments, the CAR of the present invention may include an intracellular domain. The intracellular domain may be derived from CD3zeta or a cell surface molecule selected from the group consisting of FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the co-stimulatory domain may be present. The costimulatory domain may be derived from 4-1BB (CD137) 2B4, HVEM, ICOS, LAG3, DAP10, DAP12, CD27, CD28, OX40 (CD134), CD30, CD40, ICOS (CD278), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. In one aspect the intracellular domain may be derived from CD3 Zeta SEQ ID NO. 8236. In some embodiments, the CAR may include a co-stimulatory domain derived from 4-1BB (SEQ ID NO. 8237). In some aspects, the CAR further may also include a signal sequence. The signal sequence may be derived from CD8a. In some aspects, the signal sequence may comprise the amino acid sequence of SEQ ID NO. 278.

In some embodiments, the immunotherapeutic agent may be a cytokine-cytokine receptor fusion polypeptide. The cytokine-cytokine receptor fusion polypeptide may include the whole or a portion of IL15, fused to the whole or a portion of IL15Ra to produce a IL15-IL15Ra fusion polypeptide. In one embodiment the cytokine-cytokine receptor fusion polypeptide may include amino acid sequence of SEQ ID NO. 8324 fused to the amino acid sequence of SEQ ID NO. 8324 to produce a fusion polypeptide.

In some embodiments, the effector module of the invention may be a DD-CD19 CAR fusion polypeptide. The fusion polypeptide may comprise the amino acid sequence of SEQ ID NO. 8283; 8271-8282 or 8284. In some embodiments, the effector module of the invention may be a DD-IL15-IL15Ra fusion polypeptide (SEQ ID NO. 8338; 8334-8337 or 8339-8343).

In some embodiments, the SRE may be responsive to one or more stimuli. The stimulus may be a small molecule such as but not limited to Tadalafil, Vardenafil, Sildenafil, Avanafil, Lodenafil, Mirodenafil, Udenafil, Benzamidenafil, Dasantafil, and Beminafil. In one aspect, the small molecule may be Tadalafil.

The present invention also provides a pharmaceutical composition which may include the compositions described herein and a pharmaceutically acceptable excipient.

The present invention also provides polynucleotides encoding the compositions and the pharmaceutical compositions described herein.

Also provided herein are immune cells for adoptive transfer. The immune cells may express the compositions, pharmaceutical compositions, the polynucleotides, or the vectors described herein.

The present invention also provides methods of inducing an immune response in a cell. Such methods may include administering to the cell, a therapeutically effective amount of any of the pharmaceutical composition and administering to the cell, a therapeutically effective amount of a stimulus to modulate the expression of the immunotherapeutic agent. In some embodiments, the stimulus may be a ligand. In some aspects, the immunotherapeutic agent may be capable of inducing an immune response in the cell, in response to the stimulus.

Methods of reducing tumor burden using the compositions described herein are also provided. Such methods may include administering to the subject a therapeutically effective amount of the immune cells described herein and administering to the subject a therapeutically effective amount of a stimulus. In some embodiments, the stimulus may be a ligand. In some embodiments, the stimulus may be able to modulate the expression of the immunotherapeutic agent, thereby reducing the tumor burden. In some embodiments, the ligand is Tadalafil. In some aspects the Tadalafil may be administered to the subject at a dose ranging from about 0.1 mg/kg to about 100 mg/kg body weight of the subject. In some aspects, the Tadalafil may be administered to the subject at a dose of 10 mg/kg body weight of the subject. In some aspects, the Tadalafil may be administered to the subject at a dose of 30 mg/kg body weight of the subject. In some aspects, the Tadalafil may be administered to the subject at a dose of 100 mg/kg body weight of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale; emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

FIG. 1 shows an overview diagram of a biocircuit system of the invention. The biocircuit comprises a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces a signal or outcome. The effector module comprises at least one stimulus response element (SRE) and one payload.

FIG. 2 shows representative effector modules carrying one payload. The signal sequence (SS), SRE and payload may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site. An optional linker may be inserted between each component of the effector module.

FIG. 3 shows representative effector modules carrying two payloads without a cleavage site. The two payloads may be either directly linked to each other or separated.

FIG. 4 shows representative effector modules carrying two payloads with a cleavage site. In one embodiment, an SS is positioned at the N-terminus of the construct, while other components: SRE, two payloads and the cleavage site may be located at different positions (A to L). In another embodiment, the cleavage site is positioned at the N-terminus of the construct (M to X). An optional linker may be inserted between each component of the effector module.

FIG. 5 shows effector modules of the invention carrying two payloads, where an SRE is positioned at the N-terminus of the construct (A to L), while SS, two payloads and the cleavage site can be in any configuration. An optional linker may be inserted between each component of the effector module.

FIG. 6 shows effector modules of the invention carrying two payloads, where either the two payloads (A to F) or one of the two payloads (G to X) is positioned at the N-terminus of the construct (A to L), while SS, SRE and the cleavage site can be in any configuration. An optional linker may be inserted between each component of the effector module.

FIG. 7 depicts representative configurations of the stimulus and effector module within a biocircuit system. A trans-membrane effector module is activated either by a free stimulus (A) or a membrane bound stimulus (B) which binds to SRE. The response to the stimulus causes the cleavage of the intracellular signal/payload, which activates down-stream effector/payload.

FIG. 8 depicts a dual stimulus-dual presenter biocircuit system, where two bound stimuli (A and B) from two different presenters (e.g., different cells) bind to two different effector modules in a single receiver (e.g., another single cell) simultaneously and create a dual-signal to downstream payloads.

FIG. 9 depicts a dual stimulus-single presenter biocircuit system, where two bound stimuli (A and B) from the same presenter (e.g., a single cell) bind to two different effector modules in another single cell simultaneously and create a dual-signal.

FIG. 10 depicts a single-stimulus-bridged receiver biocircuit system. In this configuration, a bound stimulus (A) binds to an effector module in the bridge cell and creates a signal to activate a payload which is a stimulus (B) for another effector module in the final receiver (e.g., another cell).

FIG. 11 depicts a single stimulus-single receiver biocircuit system, wherein the single receiver contains the two effector modules which are sequentially activated by a single stimulus.

FIG. 12 depicts a biocircuit system which requires a dual activation. In this embodiment, one stimulus must bind the transmembrane effector module first to prime the receiver cell being activated by the other stimulus. The receiver only activates when it senses both stimuli (B).

FIG. 13 depicts a standard effector module of a chimeric antigen receptor (CAR) system which comprises an antigen binding domain as an SRE, and signaling domain(s) as payload.

FIG. 14 depicts the structure design of a regulatable CAR system, where the trans-membrane effector modules comprise antigen binding domains sensing an antigen and a first switch domain and the intracellular module comprises a second switch domain and signaling domains. A stimulus (e.g., a dimerization small molecule) can dimerize the first and second switch domains and assemble an activated CAR system.

FIG. 15 shows schematic representation of CAR systems having one (A) or two (B and C) SREs incorporated into the effector module.

FIG. 16 depicts a split CAR design to control T cell activation by a dual stimulus (e.g., an antigen and small molecule). (A) shows normal T cell activation which entails a dual activation of TCR and co-stimulatory receptor. The regular CAR design (B) combines the antigen recognition domain with TCR signaling motif and co-stimulatory motif in a single molecule. The split CAR system separates the components of the regular CAR into two separate effector modules which can be reassembled when a heterodimerizing small molecule (stimulus) is present.

FIG. 17 depicts the positive and negative regulation of CAR engineered T cell activation. The absence or presence of a second stimulus can negatively (A) or positively (B) control T cell activation.

FIG. 18 shows schematic representation of gated activation of CAR engineered T cells. If a normal cell that has no stimulus (e.g., an antigen) (A) or an antigen that cannot bind to the trans-membrane effector module (B), or only an antigen that activates the trans-membrane effector module and primes the receiver T cell to express the second effector (C), the receiver T cell remains inactive. When both stimuli (e.g. two antigens) that bind the trans-membrane effector module and the primed effector, are present on the presenter cell (e.g. a cancer cell), the T cell is activated (D).

FIG. 19A shows the percent of CAR positive cells obtained with cells transduced with different volumes of virus related to the CAR constructs. FIG. 19B shows the percentage of CAR positive cells with vardenafil treatment. FIG. 19C shows the expression of the CAR with CD3/CD28 bead restimulation. FIG. 19D shows the percentage of CAR positive cells with different concentrations of virus and in the presence or absence of CD3/CD28 bead restimulation. FIG. 19E shows the dose response of CD19 CAR constructs to sildenafil, vardenafil and tadalafil. FIG. 19F shows the response of CD19 CAR constructs to different ligands and with varying the duration of ligand treatment. FIG. 19G shows IL2 and IFNγ levels obtained from the supernatants of cocultures of effector and target cells in the presence of vardenafil. FIG. 19H shows the percentage of CAR positive T cells obtained with Tadalafil treatment. FIG. 19I shows the IL2 and IFNγ levels obtained from the supernatants of cocultures of effector and target cells in the presence of tadalafil. FIG. 19J shows the target cell killing by CAR expressing T cells in the presence of vardenafil. FIG. 19K shows the proliferation of target cells cocultured with CAR expressing T cells in the presence of vardenafil.

FIG. 20A shows the total flux of Nalm6 luc cells in mice, in the presence of tadalafil. FIG. 20B and FIG. 20C show the pharmacokinetics of vardenafil and tadalafil in plasma of mice after injections of ligand at indicated doses.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description will control.

I. Introduction Protein Regulation

The ability to conditionally control protein levels is a powerful tool in gene and cell therapy. Techniques to control protein expression on a genetic level have been widely studied. The Cre-Lox technology provides a useful approach to activate or inactivate genes. Tissue or cell specific promoters can be used to control spatial and temporal expression of genes of interest. However, this system is limited in application due to the irreversible nature of the perturbation. The transcription of the gene of interest can be conditionally regulated using tools such as Doxycycline (Dox)-inducible system. Alternatively, the stability of mRNA can be regulated using RNA interference techniques. However, methods targeting DNA or RNA are slow acting, irreversible and have low efficiency.

Direct manipulation of activities at the protein level provides significant advantages in flexibility, reversibility and speed. Strategies which directly trigger a cell's natural degradation system have been developed. Szostak and colleagues showed that a small peptide sequence could be fused to the N-terminus of a protein of interest to modulate protein stability (Park, E-C., et al., Proc. Natl. Acad. Sci. U.S.A. 1992, 89:1249-1252). Varshaysky and coworkers isolated a temperature-sensitive peptide sequence that greatly reduced the half-life of dihydrofolate reductase (DHFR) at the non-permissive temperatures (Dohmen et al. Science 1994, 263:1273-1276). These mutants have been widely used to study protein functions in yeast (Labib et al. Science 2000, 288:1643-1646; and Kanemaki et al. Nature 2003, 423:720-724). Subsequently, reversible systems employing a rapamycin derivative for the regulation of GSK-3β kinase fused to an unstable triple-mutant of the FRB domain (FRB*) were developed. The rapamycin derivative induces dimerization of the FRB*-GSK-3β and endogenous FKBP12 and stabilizes the FRB* fusion thus restoring the function of the fused kinase. (Stankunas et al., Mol Cell. 2003; 12:1615-1624 and Liu et al., Nature. 2007; 446:79-82).

Building on the FRB* domain system, Banaszynski, et al., developed a cell-permeable ligand system using mutants of FKBP12 protein which were engineered to be unstable in the absence of a high-affinity ligand, Shield-1. (Banaszynski et al., Cell. 2006; 126:995-1004). They termed these unstable domains, destabilizing domains (DDs).

The FKBP/shield-1 tuning system has been successfully used in several studies to control target proteins. For example, Dettwier et al., fused FKBP to tune the express of NADPH P450 oxidoreductase (POR) (Dettwier et al., PLoS One, 2014, 9(11): e113540).

The FKBP DD-shield system has been used in cell lines, transgenic mice, protozoan Entamoeba histolytica, the flatworm Caenorhabditis elegans, the medaka, and transgenic xenografts to investigate the activity of a protein of interest (Maynard-Smith et al., J Biol Chem. 2007, 282(34): 24866-24872; Liu et al., Int J Parasitol. 2014, 44(10):729-735; Cho et al., PLoS One. 2013, 8(8): e72393); Banaszynski et al. Nat Med. 2008, 14(10):1123-1127; Rodriguez and Wolfgang, Chem Biol. 2011, 19(3):391-398; and Froschauer et al., PLoS One, 2015, 10(7): e0131252), for iPSC reprogramming (Sui et al., Stem cell Reports, 2014, 2(5): 721-733).

In addition, the destabilizing domain has been used for the conditional knock-down or knock-out of the target gene fused with the destabilizing domain. Park et al achieved this genomic engineering by CRISPR/Cas9-mediated homologous recombination and a donor template coding for a resistance cassette and the DD-tagged TCOF1 sequence (Park et al., PLoS One. 2014, 9(4): e95101).

More recently protein switches useful as biosensors as well as new chimeric antigen receptors and other small molecule stabilization frameworks have been disclosed (An W, et al. PLoS ONE, 2015, 10(12): e0145783. doi: 10.1371/journal.pone.0145783; Nicholes, et al., Protein Engineering, Design & Selection, 2016, vol. 29 no. 2, pp. 77-85; Nath, et al., Biochemical and Biophysical Research Communications, 2016, 470: 411e416); Stevers, et al., PNAS, 2016, vol. 119, no. 9, pp. E112-1161; Juillerat, A. et al., Sci. Rep. 2016, 6: 18950; Roybal, Cell, 2016, vol. 164, pp. 1-10; and Morsut, Cell, 2016, vol. 164, pp. 1-12).

One drawback of the FKBP/Shield-1 is that Shield-1 is a novel drug whose biodistribution is not fully characterized and it is not known to what extent Shield-1 crosses the blood-brain barrier.

Other DD ligand pairs include estrogen receptor domains which can be regulated by several estrogen receptor antagonists (Miyazaki et al., J Am Chem. Soc., 2012, 134(9): 3942-3945), and fluorescent destabilizing domain (FDD) derived from bilirubin-inducible fluorescent protein, UnaG. A FDD and its cognate ligand bilirubin (BR) can induce degradation of a protein fused to the FDD (Navarro et al., ACS Chem Biol., 2016, June 6, Epub). Other known DDs and their applications in protein stability include those described in U.S. Pat. Nos. 8,173,792 and 8,530,636, the contents of which are each incorporated herein by reference in their entirety.

In an orthogonal approach, the destabilizing domains of the bacterial dihydrofolate reductase (ecDHFR) were explored. (Iwamoto et al., Chem Biol. 2010, 17(9):981-988; and Tai et al., PLoS One. 2012, 7(9): e46269). Numerous inhibitors of DHFR have been developed as drugs and one such inhibitor Trimethoprim (TMP), inhibits ecDHFR much more potently than mammalian DHFR providing specificity to the interaction. Additionally, TMP is commercially available and has desirable pharmacological properties making this protein-ligand pair ideal for development for use as a biocircuit (Iwamoto, et al., Chem Biol. (2010) Sep. 24; 17(9): 981-988).

The present invention provides novel protein domains displaying small molecule dependent stability. Such protein domains are called destabilizing domains (DDs). In the absence of its binding ligand, the DD is destabilizing and causes degradation of a payload fused to the DD (e.g., a protein of interest (POI), while in the presence of its binding ligand, the fused DD and payload can be stabilized and its stability is dose dependent. Methods for tuning the expression level and activity of a protein of interest using the DDs, effector modules, biocircuit systems and compositions of the invention are also provided. In some embodiments, the SRE may be a destabilizing domain (DD). In some examples, the DD may be a portion or region of human protein PDE5. In this context, the biocircuit system is a DD biocircuit system.

The present invention expands upon the technology of tuning protein stability using novel destabilizing domains derived from human PDE5 protein. The destabilization and stabilization of a protein of interest, e.g., a transgene for gene therapy, can be controlled by PDE5 mutant DDs having destabilizing or stabilizing properties and their ligands, e.g. Sildenafil and Vardenafil specifically binding to such protein domains. The presence and/or absence of a small molecule ligand can tune the activity of a payload (e.g., a protein of interest) that is operably linked to the destabilizing domain.

Immunotherapy

Cancer immunotherapy aims' at the induction or restoration of the reactivity of the immune system towards cancer. Significant advances in immunotherapy research have led to the development of various strategies which may broadly be classified into active immunotherapy and passive immunotherapy. In general, these strategies may be utilized to directly kill cancer cells or to counter the immunosuppressive tumor microenvironment. Active immunotherapy aims at induction of an endogenous, long-lasting tumor-antigen specific immune response. The response can further be enhanced by non-specific stimulation of immune response modifiers such as cytokines. In contrast, passive immunotherapy includes approaches where immune effector molecules such as tumor-antigen specific cytotoxic T cells or antibodies are administered to the host. This approach is short lived and requires multiple applications.

Despite significant advances, the efficacy of current immunotherapy strategies is limited by associated toxicities. These are often related to the narrow therapeutic window associated with immunotherapy, which in part, emerges from the need to push therapy dose to the edge of potentially fatal toxicity to get a clinically meaningful treatment effect. Further, dose expands in vivo since adoptively transferred immune cells continue to proliferate within the patient, often unpredictably.

A major risk involved in immunotherapy is the on-target but off tumor side effects resulting from T-cell activation in response to normal tissue expression of the tumor associated antigen (TAA). Clinical trials utilizing T cells expressing T-cell receptor against specific TAA reported skin rash, colitis and hearing loss in response to immunotherapy.

Immunotherapy may also produce on target, on-tumor toxicities that emerge when tumor cells are killed in response to the immunotherapy. The adverse effects include tumor lysis syndrome, cytokine release syndrome and the related macrophage activation syndrome. Importantly, these adverse effects may occur during the destruction of tumors, and thus even a successful on-tumor immunotherapy might result in toxicity. Approaches to regulatably control immunotherapy are thus highly desirable since they have the potential to reduce toxicity and maximize efficacy.

The present invention provides systems, compositions, immunotherapeutic agents and methods for cancer immunotherapy. These compositions provide tunable regulation of gene expression and function in immunotherapy. The present invention also provides biocircuit systems, effector modules, stimulus response elements (SREs) and payloads, as well as polynucleotides encoding any of the foregoing. In one aspect, the systems, compositions, immunotherapeutic agents and other components of the invention can be controlled by a separately added stimulus, which provides a significant flexibility to regulate cancer immunotherapy. Further, the systems, compositions and the methods of the present invention may also be combined with therapeutic agents such as chemotherapeutic agents, small molecules, gene therapy, and antibodies.

The tunable nature of the systems and compositions of the invention has the potential to improve the potency and duration of the efficacy of immunotherapies. Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present invention allows maximizing the potential of cell therapy without irretrievably killing and terminating the therapy.

In particular, present invention provides methods for fine tuning of immunotherapy after administration to patients. This in turn improves the safety and efficacy of immunotherapy and increases the subject population that may benefit from immunotherapy.

II. Compositions of the Invention

A variety of strategies that can directly control protein, e.g., a transgene, expression and function are available. The present invention provides novel protein domains displaying small molecule dependent stability. Such protein domains are called destabilizing domains (DDs). In the absence of its binding ligand, the DD causes degradation of a payload such as a protein of interest (POI) that is operably linked to the DD, while in the presence of its binding ligand, the fused DD and payload can be stabilized and its stability is dose dependent.

According to the present invention, novel destabilizing domains derived from human hPDE5 (cGMP-specific phosphodiesterase type 5; also referred to as cGMP-specific 3′,5′-cyclic phosphodiesterase) protein are provided. The destabilizing domain (DD) mutants are derived from the human PDE5 protein, comprising the amino acid sequence of SEQ ID NO. 1 (encoded by the nucleic acid sequence of SEQ ID NO. 2). The hPDE5 DD mutant may also comprise more than one mutation in the catalytic domain of human PDE5 (SEQ ID NO. 3), encoded by nucleic acid sequence of SEQ ID NO. 339, e.g., two, three, four, five or more mutations. These hPDE5 DDs can bind to Sildenafil and/or Vardenafil and be stabilized. In some embodiments, the hPDE5 DD may include a methionine appended to the N terminus of catalytic domain (SEQ ID NO. 237), encoded by SEQ ID NO. 4.

According to the present invention, biocircuit systems are provided which comprise, at their core, at least one effector module system. Such effector module systems comprise at least one effector module having associated, or integral therewith, one or more stimulus response elements (SREs). The overall architecture of a biocircuit system of the invention is illustrated in FIG. 1 . In general, a stimulus response element (SRE) may be operably linked to a payload construct which could be any protein of interest (POI) (e.g., an immunotherapeutic agent), to form an effector module. The SRE, when activated by a particular stimulus, e.g., a small molecule, can produce a signal or outcome, to regulate transcription and/or protein levels of the linked payload either up or down by perpetuating a stabilizing signal or destabilizing signal, or any other types of regulation. A much-detailed description of a biocircuit system can be found in co-owned U.S. Provisional Patent Application No. 62/320,864 filed Apr. 11, 2016, 62/466,596 filed Mar. 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety). In accordance with the present invention, biocircuit systems, effector modules, SREs and components that tune expression levels and activities of any agents used for immunotherapy are provided. In particular, biocircuit systems and effector modules comprising the novel hPDE5 destabilizing domains discussed herein are provided.

As used herein, a “biocircuit” or “biocircuit system” is defined as a circuit within or useful in biologic systems comprising a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces at least one signal or outcome within, between, as an indicator of, or on a biologic system. Biologic systems are generally understood to be any cell, tissue, organ, organ system or organism, whether animal, plant, fungi, bacterial, or viral. It is also understood that biocircuits may be artificial circuits which employ the stimuli or effector modules taught by the present invention and effect signals or outcomes in acellular environments such as with diagnostic, reporter systems, devices, assays or kits. The artificial circuits may be associated with one or more electronic, magnetic, or radioactive components or parts.

In accordance with the present invention, a biocircuit system may be a destabilizing domain (DD) biocircuit system, a dimerization biocircuit system, a receptor biocircuit system, and a cell biocircuit system. Any of these systems may act as a signal to any other of these biocircuit systems. In some embodiments, the present invention provides biocircuit systems, effector modules and compositions comprising the DDs of the present invention. In one aspect, the biocircuit system is a DD biocircuit system.

In one aspect of the present invention, the biocircuit system is a DD biocircuit system. The DD is a hPDE5 derived DD.

Effector Modules and SREs for Immunotherapy

In accordance with the present invention, biocircuit systems, effector modules, SREs, and components that tune expression levels and activities of any agents used for immunotherapy are provided. As non-limiting examples, an immunotherapeutic agent may be an antibody and fragments and variants thereof, a cancer specific T cell receptor (TCR) and variants thereof, an anti-tumor specific chimeric antigen receptor (CAR), a chimeric switch receptor, an inhibitor of a co-inhibitory receptor or ligand, an agonist of a co-stimulatory receptor and ligand, a cytokine, chemokine, a cytokine receptor, a chemokine receptor, a soluble growth factor, a metabolic factor, a suicide gene, a homing receptor, or any agent that induces an immune response in a cell and a subject.

As stated, the biocircuits of the invention include at least one effector module as a component of an effector module system. As used herein, an “effector module” is a single or multi-component construct or complex comprising at least (a) one or more stimulus response elements (SREs) and (b) one or more payloads (i.e. proteins of interest (POIs)). In the context of the present invention, the SRE is a DD derived from human PDE5 protein.

As used herein a “stimulus response element (SRE)” is a component of an effector module which is joined, attached, linked to or associated with one or more payloads of the effector module and in some instances, is responsible for the responsive nature of the effector module to one or more stimuli. As used herein, the “responsive” nature of an SRE to a stimulus may be characterized by a covalent or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus. Further, the response of any SRE to a stimulus may be a matter of degree or kind. The response may be a partial response. The response may be a reversible response. The response may ultimately lead to a regulated signal or output. Such output signal may be of a relative nature to the stimulus, e.g., producing a modulatory effect of between 1% and 100% or a factored increase or decrease such as 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more.

In some embodiments, the biocircuit system of the present invention comprising a stimulus and an effector module of the invention. The DD of the effector module binds to the stimulus and regulates the stability of the linked payload. The DD may destabilize the protein of interest by a destabilization ratio between 0, and 0.09, wherein the destabilization ratio comprises the ratio of expression, function or level of a protein of interest in the absence of the stimulus specific to the DD to the expression, function or level of the protein of interest that is expressed constitutively, and in the absence of the stimulus specific to the DD. In some embodiments, the DD may stabilize the protein of interest by a stabilization ratio of 1 or more, wherein the stabilization ratio comprises the ratio of expression, function or level of a protein of interest in the presence of the stimulus to the expression, function or level of the protein of interest in the absence of the stimulus.

In some embodiments, the present invention provides methods for modulating protein expression, function or level. In some aspects, the modulation of protein expression, function or level refers to modulation of expression, function or level by at least about 20%, such as by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.

According to the present invention, biocircuit systems and effector modules of the invention can be used to regulate the expression and activity of a payload in response to the presence or absence of a ligand that specifically binds to the DD integrated within the biocircuit system and effector module.

In some aspects, DDs, effector modules and biocircuit systems of the invention may be used to regulate the expression, function and activity of a payload in a cell or a subject. The regulation refers to a level of change of its expression, function and activity, by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.

In some embodiments, the mutation co-efficient of the biocircuits of the invention may also be measured as a way of evaluating the ability of the DD to modulate protein expression, function or level. As used herein, the “mutation co-efficient” refers to the expression, function or level or a protein of interest, appended to a particular DD mutant, in the absence of the stimulus specific to the SRE; to the protein expression, function or level of the protein of interest, appended to the corresponding wildtype sequence from which the particular DD mutant is derived and in the absence of the stimulus. The mutation co-efficient is indicative of the contribution of the destabilizing mutations towards the basal expression of the protein independent of the whether the corresponding wildtype protein can be destabilized without any mutations. In some aspects, the mutation co-efficient ratio is at least 0, such as by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at least, 0-0.1, 0-0.2, 0-0.3, 0-0.4, 0-0.5, 0-0.6, 0-0.7, 0-0.8, 0-0.9, 0.1-0.2, 0.1-0.3, 0.1-0.4, 0.1-0.5, 0.1-0.6, 0.1-0.7, 0.1-0.8, 0.1-0.9, 0.2-0.3, 0.2-0.4, 0.2-0.5, 0.2-0.6, 0.2-0.7, 0.2-0.8, 0.2-0.9, 0.3-0.4, 0.3-0.5, 0.3-0.6, 0.3-0.7, 0.3-0.8, 0.3-0.9, 0.4-0.5, 0.4-0.6, 0.4-0.7, 0.4-0.8, 0.4-0.9, 0.5-0.6, 0.5-0.7, 0.5-0.8, 0.5-0.9, 0.6-0.7, 0.6-0.8, 0.6-0.9, 0.7-0.8, 0.7-0.9 or 0.8-0.9.

In some embodiments, the present invention provides methods for modulating protein, expression, function or level by measuring the stabilization ratio, destabilization ratio, and/or destabilizing mutation co-efficient. As used herein, the “stabilization ratio” is the ratio of expression, function or level of a protein of interest in response to the stimulus to the expression, function or level of the protein of interest in the absence of the stimulus specific to the SRE. In some aspects, the stabilization ratio is at least 1, such as by at least 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-95, 20-100, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-95, 30-100, 40-50, 40-60, 40-70, 40-80, 40-90, 40-95, 40-100, 50-60, 50-70, 50-80, 50-90, 50-95, 50-100, 60-70, 60-80, 60-90, 60-95, 60-100, 70-80, 70-90, 70-95, 70-100, 80-90, 80-95, 80-100, 90-95, 90-100 or 95-100. As used herein, the “destabilization ratio” is the ratio of expression, function or level of a protein of interest in the absence of the stimulus specific to the effector module to the expression, function or level of the protein of interest, that is expressed constitutively and in the absence of the stimulus specific to the SRE. As used herein “constitutively” refers to the expression, function or level a protein of interest that is not linked to an SRE, and is therefore expressed both in the presence and absence of the stimulus to the SRE. As used herein, the “destabilizing mutation co-efficient” may be defined as the ratio of expression or level of a protein of interest that is appended to a DD, in the absence of the stimulus specific to the effector module to the expression, function or level of the protein that is appended to the wild type protein from which the DD is derived. In some aspects, the destabilization ratio and/or the destabilizing mutation co-efficient is at least 0, such as by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at least, 0-0.1, 0-0.2, 0-0.3, 0-0.4, 0-0.5, 0-0.6, 0-0.7, 0-0.8, 0-0.9, 0.1-0.2, 0.1-0.3, 0.1-0.4, 0.1-0.5, 0.1-0.6, 0.1-0.7, 0.1-0.8, 0.1-0.9, 0.2-0.3, 0.2-0.4, 0.2-0.5, 0.2-0.6, 0.2-0.7, 0.2-0.8, 0.2-0.9, 0.3-0.4, 0.3-0.5, 0.3-0.6, 0.3-0.7, 0.3-0.8, 0.3-0.9, 0.4-0.5, 0.4-0.6, 0.4-0.7, 0.4-0.8, 0.4-0.9, 0.5-0.6, 0.5-0.7, 0.5-0.8, 0.5-0.9, 0.6-0.7, 0.6-0.8, 0.6-0.9, 0.7-0.8, 0.7-0.9 or 0.8-0.9.

The SRE of the effector module may be selected from, but is not limited to, a peptide, peptide complex, peptide-protein complex, protein, fusion protein, protein complex, protein-protein complex. The SRE may comprise one or more regions derived from any natural or mutated protein, or antibody. In this aspect, the SRE is an element, when responding to a stimulus, can tune intracellular localization, intramolecular activation, and/or degradation of payloads.

In some embodiments, effector modules of the present invention may comprise additional features that facilitate the expression and regulation of the effector module, such as one or more signal sequences (SSs), one or more cleavage and/or processing sites, one or more targeting and/or penetrating peptides, one or more tags, and/or one or more linkers. Additionally, effector modules of the present invention may further comprise other regulatory moieties such as inducible promoters, enhancer sequences, microRNA sites, and/or microRNA targeting sites. Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature. For example, an SRE may represent a destabilizing domain, while mutations in the protein payload may alter its cleavage sites or dimerization properties or half-life and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features. Consequently, the present invention embraces biocircuits which are multifactorial in their tenability. Such biocircuits may be engineered to contain one, two, three, four or more tuned features.

As shown in FIG. 2 , representative effector module embodiments comprising one payload, i.e. one immunotherapeutic agent are illustrated. Each components of the effector module may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site. An optional linker may be inserted between each component of the effector module.

FIGS. 3 to 6 illustrate representative effector module embodiments comprising two payloads, i.e. two immunotherapeutic agents. In some aspects, more than two immunotherapeutic agents (payloads) may be included in the effector module under the regulation of the same SRE (e.g., the same DD). The two or more agents may be either directly linked to each other or separated (FIG. 3 ). The SRE may be positioned at the N-terminus of the construct, or the C-terminus of the construct, or in the internal location.

In some aspects, the two or more immunotherapeutic agents may be the same type such as two antibodies, or different types such as a CAR construct and a cytokine IL12. Biocircuits and components utilizing such effector molecules are given in FIGS. 7-12 .

As used herein a “payload” or “target payload” is defined as any protein or nucleic acid whose function is to be altered. Payloads may include any coding or non-coding gene or any protein or fragment thereof, or fusion constructs, or antibodies.

Payloads are often associated with one or more SREs (e.g., DDs) and may be encoded alone or in combination with one or more DD in a polynucleotide of the invention. Payloads themselves may be altered (at the protein or nucleic acid level) thereby providing for an added layer of tenability of the effector module. For example, payloads may be engineered or designed to contain mutations, single or multiple, which affect the stability of the payload or its susceptibility to degradation, cleavage or trafficking. The combination of a DD which can have a spectrum of responses to a stimulus with a payload which is altered to exhibit a variety of responses or gradations of output signals, e.g., expression levels, produce biocircuits which are superior to those in the art. For example, mutations or substitutional designs such as those created for IL12 in WO2016048903 (specifically in Example 1 therein), the contents of which are incorporated herein by reference in their entirety, may be used in any protein payload in conjunction with a DD of the present invention to create dual tunable biocircuits. The ability to independently tune both the DD and the payload greatly increases the scope of uses of the effector modules of the present invention.

Effector modules may be designed to include one or more payloads, one or more DDs, one or more cleavage sites, one or more signal sequences, one or more tags, one or more targeting peptides, and one or more additional features including the presence or absence of one or more linkers. Representative effector module embodiments of the invention are illustrated in FIGS. 2-6 . In some aspects, the DD can be positioned at the N-terminal end, or the C-terminal end, or internal of the effector module construct. Different components of an effector module such as DDs, payloads and additional features are organized linearly in one construct, or are separately constructed in separate constructs.

Additionally, effector modules of the present invention may further comprise other regulatory moieties such as inducible promoters, enhancer sequences, microRNA sites, and/or microRNA targeting sites that provide flexibility on controlling the activity of the payload. The payloads of the present invention may be any natural proteins and their variants, or fusion polypeptides, antibodies and variants thereof, transgenes and therapeutic agents.

The stimulus of the biocircuit system may be, but is not limited to, a ligand, a small molecule, an environmental signal (e.g., pH, temperature, light and subcellular location), a peptide or a metabolite. In one aspect of the present invention, the stimulus is a hPDE5 DD binding ligand including Sildenafil and Vardenafil.

Polypeptides of DDs, biocircuit systems and effector modules comprising such DDs and payload constructs, other components, polynucleotides encoding these polypeptides and variants thereof, vectors comprising these polynucleotides, are provided in the present invention. The vector may be a plasmid or a viral vector including but not limited to a lentiviral vector, a retroviral vector, a recombinant AAV vector and oncolytic viral vector.

The position of the payload with respect to the DD, within the SRE may be varied to achieve optimal DD regulation. In some embodiments, the payload may be fused to the N terminus of the DD. In another embodiment, the payload may be fused to the C terminus of the DDs. An optional start codon nucleotide sequence encoding for methionine may be added to the DD and/or payload.

In some embodiments, more than one biocircuit system may be used in combination to control various protein functions in the same cell or organism, each of which uses different DD and ligand pair and can be regulated separately.

In some embodiments, biocircuits of the invention may be modified to reduce their immunogenicity. Immunogenicity is the result of a complex series of responses to a substance that is perceived as foreign and may include the production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity responses, and anaphylaxis. Several factors can contribute to protein immunogenicity, including, but not limited to protein sequence, route and frequency of administration and patient population. In a preferred embodiment, protein engineering may be used to reduce the immunogenicity of the compositions of the invention. In some embodiments, modifications to reduce immunogenicity may include those that reduce binding of the processed peptides derived from the sequence of the compositions of the invention, to the MHC proteins. For example, amino acid may be modified such that virtually none or a minimal of number of immune epitopes predicted to bind to any prevalent MHC alleles are present in the compositions of the invention. Several methods to identify MHC binding epitopes of known protein sequences are known in the art and may be used to score epitopes in the compositions of the present invention. Such methods are disclosed in US Patent Publication No. US20020119492, US20040230380, and US 20060148009; the contents of each of which are incorporated by reference in their entirety.

Epitope identification and subsequent sequence modification may be applied to reduce immunogenicity. The identification of immunogenic epitopes may be achieved either physically or computationally. Physical methods of epitope identification may include, for example, mass spectrometry and tissue culture/cellular techniques. Computational approaches that utilize information related to antigen processing, loading and display, structural and/or proteomic data for identifying peptides that may result from antigen processing, and that are likely to have good binding characteristics in the groove of the MHC may also be utilized. One or more mutations may be introduced into the biocircuits of the invention to render the identified epitope less or non-immunogenic, while maintaining functionality.

In some embodiments, protein modifications engineered into the structure of the compositions of the invention to interfere with antigen processing and peptide loading such as glycosylation and PEGylation, may also be useful in the present invention. Compositions of the invention may also be engineered to include non-classical amino acid sidechains. Any of the methods discussed in International Patent Publication No. WO2005051975 for reducing immunogenicity may be useful in the present invention (the contents of which are incorporated by reference in their entirety).

In one embodiment, patients may also be stratified according to the immunogenic peptides presented by their immune cells and may be utilized as a parameter to determine patient cohorts that may therapeutically benefit from the compositions of the invention.

In some embodiments, reduced immunogenicity may be achieved by limiting immunoproteasome processing. The proteasome is an important cellular protease that is found in two forms: the constitutive proteasome, which is expressed in all cell types and which contains catalytic subunits and the immunoproteasome that is expressed in cells of the hematopoietic lineage, and which contains different active subunits termed low molecular weight proteins (LMP) namely LMP-2, LMP-7 and LMP-10. Immunoproteasomes exhibit altered peptidase activities and cleavage site preferences that result in more efficient liberation of many MEW class I epitopes. A well described function of the immunoproteasome is to generate peptides with hydrophobic C terminus that can be processed to fit in the groove of MEW class I molecules. Deol P et al. have shown that immunoproteasomes may lead to a frequent cleavage of specific peptide bonds and thereby to a faster appearance of a certain peptide on the surface of the antigen presenting cells; and enhanced peptide quantities (Deol P et al. (2007) J Immunol 178 (12) 7557-7562; the contents of which are incorporated herein reference in its entirety). This study indicates that reduced immunoproteasome processing may be accompanied by reduced immunogenicity. In some embodiments, immunogenicity of the compositions of the invention may be reduced by modifying the sequence encoding the compositions of the invention to prevent immunoproteasome processing. Biocircuits of the present invention may also be combined with immunoproteasome-selective inhibitors to achieve the same effects. Examples of inhibitors useful in the present invention include UK-101 (Bli selective compound), IPSI-001, ONX 0914 (PR-957), and PR-924 (IPSI).

In some embodiments, effector modules of the present invention may include one or more degrons to tune expression. As used herein, a “degron” refers to a minimal sequence within a protein that is sufficient for the recognition and the degradation by the proteolytic system. An important property of degrons is that they are transferrable, that is, appending a degron to a sequence confers degradation upon the sequence. In some embodiments, the degron may be appended to the destabilizing domains, the payload or both. Incorporation of the degron within the effector module of the invention, confers additional protein instability to the effector module and may be used to reduce basal expression. In some embodiments, the degron may be an N-degron, a phospho degron, a heat inducible degron, a photosensitive degron, an oxygen dependent degron. As a non-limiting example, the degron may be an Ornithine decarboxylase degron as described by Takeuchi et al. (Takeuchi J et al. (2008). Biochem J. 2008 Mar. 1; 410(2):401-7; the contents of which are incorporated by reference in their entirety). Other examples of degrons useful in the present invention include degrons described in International patent publication Nos. WO2017004022, WO2016210343, and WO2011062962; the contents of each of which are incorporated by reference in their entirety.

In some embodiments, the effector modules of the present invention may include degrons at their C termini. The degrons may comprise -GG, -RG, -KG, -QG, -WG, -PG, and -AG as the penultimate and the ultimate amino acids of the SREs. Furthermore, certain −2 amino acids (D, E, V, I and L) may be more enriched in the C terminus of the of the effector modules. Other degrons include, but are not limited, to RxxG motif, wherein x is any amino acid, C-terminal twin glutamic acid (EE) motif, and motifs that comprise an arginine at the −3 positions. Degrons may also be selected from the R-3 motif, G-end, R at −3, A-end, A at −2, V at −2 positions. Any of the degrons described in Koren et al., 2018, Cell 173, 1-14, may be useful in the present invention (the contents of which are incorporated by reference in their entirety). In some aspects, the expression of the effector module may be tuned by altering its overall amino acid composition. In some aspects, the amino acid composition of the effector module may be tuned to reduce basal expression. In some embodiments, basal expression may be tuned by increasing the number of bulky aromatic residues such as tryptophan (W), phenylalanine (F), and tyrosine (Y) in the effector module. Such bulky amino acids are known to reduce protein stability. In some embodiments, the amino acid composition of the SREs may be enriched with acidic residues such as, but not limited to, aspartic acid (D) and glutamic acid (E), and positively charged lysine (K), if an increase in the basal expression of the SRE is desired.

In some embodiments, the endoplasmic reticulum associated degradation (ERAD) pathway may be used to optimize degradation of the payloads described herein e.g. secreted and membrane cargos. In one embodiment, the effector modules of the invention may be directed to the ER E3 ligases by using adaptor proteins or protein domains. The endoplasmic reticulum is endowed with a specialized machinery to ensure proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by the ERAD pathway, a process through which folding defective proteins are selected and ultimately degraded by the ubiquitin proteasome system. ERAD proceeds through four main steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasome degradation. Any of these steps may be modulated to optimize the degradation of the payloads and the effector modules described herein. Protein adaptors within the ER membrane, link substrate recognition to the ERAD machinery (herein referred to as the “dislocon”), which causes the dislocation of the proteins from the ER. Non-limiting examples of protein adaptors that may be used to optimize ERAD pathway degradation include, but are not limited to SEL1L (an adaptor that links glycan recognition to the dislocon), Erlins (intermembrane substrate adaptors), Insigs (client specific adaptors), F-Box proteins (act as adaptors for dislocated glycoproteins in the cytoplasm) and viral-encoded adaptors.

According to the present invention, novel destabilizing domains derived from human hPDE5 (cGMP-specific phosphodiesterase type 5) protein are provided. The destabilizing mutants are derived from the human PDE5 protein, comprising the amino acid sequence of SEQ ID NO. 1 (encoded by the nucleic acid sequence of SEQ ID NO: 2). The hPDE5 DD mutant may also comprise more than one mutation in the catalytic domain of human PDE5 of SEQ ID NO. 3 (encoded by nucleic acid sequence of SEQ ID NO. 339), e.g., two, three, four, five or more mutations. These hPDE5 DDs can bind to Sildenafil and/or Vardenafil and be stabilized.

Destabilizing Domains (DDs)

As used herein, the term “destabilizing domains (DDs)” refers to protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. Destabilizing domains (DDs) can be appended to a target protein of interest (POI) and can convey its destabilizing property to the protein of interest, causing protein degradation. The presence, absence or an amount of a small molecule ligand that binds to or interacts with the DD, can, upon such binding or interaction modulate the stability of the payload(s) and consequently the function of the payload. A protein domain with destabilizing property (e.g. a DD) is used in conjunction with a cell-permeable ligand to regulate any protein of interest when it is fused with the destabilizing domain. DDs render the attached protein of interest unstable in the absence of a DD-binding ligand such that the protein is rapidly degraded by the ubiquitin-proteasome system of the cell. However, when a specific small molecule ligand binds its intended DD as a ligand binding partner, the instability is reversed and protein function is restored. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable substrate for degradation. Moreover, its dependency on the concentration of its ligand further provides tunable control of degradation rates. Depending on the degree of binding and/or interaction the altered function of the payload may vary, hence providing a “tuning” of the payload function.

Due to its reversibility, specificity and the fast and easy regulation on protein level, the post-transcriptional tuning system provides a useful system for gene regulation. Furthermore, the regulation may be dose-dependent, thereby altering the protein-turnover rate to transform a short-lived or no detectable protein into a protein that functions for a precisely controlled period of time (Iwamoto et al., Chem. Biol. 2010, 17: 981-988).

In some embodiments, the desired characteristics of the DDs may include, but are not limited to, low protein levels in the absence of a ligand of the DD (i.e. low basal stability), large dynamic range, robust and predictable dose-response behavior, and rapid kinetics of degradation. Candidate DDs that bind to a desired ligand but not endogenous molecules may be preferred.

Candidate destabilizing domain sequence identified from protein domains of known wildtype proteins (as a template) may be mutated to generate libraries of mutants based on the template candidate domain sequence. Mutagenesis strategies used to generate DD libraries may include site-directed mutagenesis e.g. by using structure guided information, or random mutagenesis e.g. using error-prone PCR, or a combination of both. In some embodiments, destabilizing domains identified using random mutagenesis may be used to identify structural properties of the candidate DDs that may be required for destabilization, which may then be used to further generate libraries of mutations using site directed mutagenesis.

In some embodiments, novel DDs may be identified by mutating one or more amino acids in the candidate destabilizing domain to an amino acid that is vicinal to the mutation site. As used herein a vicinal amino acid refers to an amino acid that is located 1, 2, 3, 4, 5 or more amino acids upstream or downstream of the mutation site in the linear sequence and/or the crystal structure of the candidate destabilizing domain. In some embodiments, the vicinal amino acid may be a conserved amino acid (with similar physicochemical properties as the amino acid at the mutation site), a semi-conserved amino acid (e.g. negatively to positively charge amino acid) or a non-conserved amino acid (with different physicochemical properties than the amino acid at the mutation site).

In some embodiments, DD mutant libraries may be screened for mutations with altered, preferably higher binding affinity to the ligand, as compared to the wild type protein. DD libraries may also be screened using two or more ligands and DD mutations that are stabilized by some ligands but not others may be preferentially selected. DD mutations that bind preferentially to the ligand compared to a naturally occurring protein may also be selected. Such methods may be used to optimize ligand selection and ligand binding affinity of the DD. Additionally, such approaches can be used to minimize deleterious effects caused by off-target ligand binding.

In some embodiments, suitable DDs may be identified by screening mutant libraries using barcodes. Such methods may be used to detect, identify and quantify individual mutant clones within the heterogeneous mutant library. Each DD mutant within the library may have distinct barcode sequences (with respect to each other). In other instances, the polynucleotides can also have different barcode sequences with respect to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid bases. Each DD mutant within the library may also comprise a plurality of barcode sequences. When used in plurality barcodes may be used such that each barcode is unique to any other barcode. Alternatively, each barcode used may not be unique, but the combination of barcodes used may create a unique sequence that can be individually tracked. The barcode sequence may be placed upstream of the SRE, downstream of the SRE, or in some instances may be placed within the SRE. DD mutants may be identified by barcodes using sequencing approaches such as Sanger sequencing, and next generation sequencing, but also by polymerase chain reaction and quantitative polymerase chain reaction. In some embodiments, polymerase chain reaction primers that amplify a different size product for each barcode may be used to identify each barcode on an agarose gel. In other instances, each barcode may have a unique quantitative polymerase chain reaction probe sequence that enables targeted amplification of each barcode.

Inventors of the present invention investigated several human proteins and identified novel human DDs which can confer its instability features to the fused payload and facilitate the rapid degradation of the fusion polypeptide in the absence of its ligand but stabilize the fused payload in response to the binding to its ligand. Specifically, the new DDs are derived from human PDE5 protein.

Human PDE5 Mutants

In some embodiments, DDs of the invention may be derived from human cGMP-specific phosphodiesterase type 5 (PDE5); gene name: PDE5A (herein referred to as “hPDE5”). hPDE5 (phosphodiesterase 5) is a member of the 3,5′-cyclic nucleotide phosphodiesterase family which degrades/hydrolyzes cyclic GMP (cGMP) into its inactive form, GMP, and regulates cGMP signaling. The cGMP/PDE 5 signaling contributes to the development of hypertension in the vasculature, the central nervous system, and the kidney. hPDE5 is known to bind to small molecule such as Sildenafil, Vardenafil, Tadalafil, Avanafil, Lodenafil, Mirodenafil, Udenafil, Benzamidenafil, Dasantafil, Beminafil, SLx-2101, LAS 34179, UK-343,664, UK-357903, UK-371800, and BMS-341400. Sildenafil, Vardenafil, Avanafil, and Tadalafil are hPDE5 inhibitors clinically approved for the treatment of erectile dysfunction. In particular, the binding of these small molecules to hPDE5 occurs within the catalytic domain. In some embodiments, the DDs of the invention may be derived from the catalytic domain of hPDE5 (SEQ ID NO. 3) which includes of residues 535 to 860 of hPDE5 (SEQ ID NO. 1; GenBank Access NO. 076074.2) which may be stabilized by ligands such as small molecule inhibitors of hPDE5 e.g. Sildenafil and Vardenafil. As used herein the term “PDE5 WT” or “hPDE5 WT”, refers to the human wildtype PDE5 protein sequence, which is defined as SEQ ID NO. 1, with the GenBank Access NO. 076074.2. In some embodiments, DDs of the present invention may be identified by utilizing a cocktail of hPDE5 inhibitors. In other instances, the suitable DDs may be identified by screening first with one hPDE5 inhibitor and subsequently screening with a second hPDE5 inhibitor.

In one embodiment, the hPDE5 derived DD may comprise the amino acid sequence of UniProt ID: O76074 (SEQ ID NO. 1) or a portion or a fragment thereof. In another embodiment, the hPDE5 derived DD may comprise the catalytic domain of UniProt ID: O76074, spanning from amino acid position 535 to position 860 (SEQ ID NO. 3); encoded by SEQ ID NO. 339. In addition to the catalytic domain, hPDE5 derived DDs may also comprise one or more GAF domains and/or the C terminal portion that extends beyond the catalytic domain. In some embodiment, the hPDE5 derived DD may be identified by testing constructs generated by truncating the 5′ and/or the 3′ end of hPDE5. In one embodiment, the hPDE5 derived DD may be truncated and the smallest hPDE5 based DD may be identified. In another embodiment, the hPDE5 derived DD may include amino acids from position 535 to 836 of SEQ ID NO. 1 which removes the C terminal helix. In another embodiment, the hPDE5 derived DD may consist of amino acids from position 567 to 860 of UniProt ID: O76074 (SEQ ID NO. 1), or position 590 to 860 of UniProt ID: O76074 (SEQ ID NO. 1), which removes a portion of the N terminal domain. In another embodiment, the hPDE5 derived DD may consist of amino acids from position 590 to 836 of UniProt ID: O76074 (SEQ ID NO. 1), which removes a portion of the N terminal domain and the C terminal helix. The DD may include amino acids from position 535 to position 875 of UniProt ID: O76074 (SEQ ID NO. 1). In another embodiment, the hPDE5 derived DD may consist of amino acids from position 466 to 875 of UniProt ID: O76074 (SEQ ID NO. 1) or position 420 to 875 of UniProt ID: O76074 (SEQ ID NO. 1).

According to the present invention, several hPDE5 destabilizing mutations were discovered by site directed mutagenesis of the catalytic domain of wildtype human PDE5 using site directed mutagenesis. The destabilization of the mutants in the absence of its binding ligands is tested. Binding to hPDE5 ligands, Sildenafil, Tadalafil and Vardenafil to human PDE5 was tested and ligand dependent stabilization was characterized. Based on the structural analysis of hPDE5 bound to Sildenafil, several residues were selected for mutagenesis. In some embodiments one or more of the residues described herein may be mutagenized to obtain hPDE5 derived DDs. The tryptophan at position 853 of SEQ ID NO. 1 may be mutated to phenylalanine to induce hydrophobic packing near binding site, while maintaining pi bond with the nearby tryptophan at position 772. The isoleucine at position 821 may be mutated to valine or alanine, which results in hydrophobic packing near binding site. The tyrosine at position 829 may be mutated to isoleucine, valine or alanine, which results in hydrophobic packing near binding site. The aspartate at position 656 may be mutated to asparagine or leucine to break up the charge and the salt bridge on loop/helix. The tyrosine at position 728 may be mutated to phenylalanine or leucine to break the salt bridges at position 732 and to affect hydrophobic packing. Alternatively, the arginine at position 732 may be mutated to lysine or leucine to generate the inverse of the effects of mutating tyrosine at position 728. The methionine at position 625 may be mutated to leucine or isoleucine to alter packing in away from the binding site. In some embodiments, destabilizing mutations that do not affect ligand binding may be preferentially selected.

In some embodiments, new destabilizing domains of the present invention are derived from the catalytic domain of human PDE5 protein comprising the amino acid sequence of SEQ ID NO. 3. In some aspects, the destabilizing mutant domain may comprise one, two, three, four, five or more mutations such as, but not limited to, M625I, D656L, Y728L, R732L, F736A, F787A, I821A, Y829A and W853F. The DDs of the invention may further include additional mutations e.g. E535D, E536G, Q541R, K555R, S560G, F559L, F561L, F564L, F564S, S766F, V585A, N587S, K591E, I599V, K604E, K608E, N609H, K630R, K633E, N636S, I648V, N661S, S663P, L675P, Y676D, Y676N, C677R, H678R, D687A, T711A, T712S, D724N, L738H, N742S, F744L, L746S, F755L, A762S, D764V, D764N, D764G, K795E, L797F, I799T, L804P, T802P, S815C, M816A, M816T, I824T, C839S, F840S, and K852E. In some embodiments, any of the mutation sites disclosed herein may be mutated to any of the known amino acids such as Histidine, Alanine, Isoleucine, Arginine, Leucine, Aspartic acid, Lysine, Cysteine, Methionine, Glutamic acid, Phenylalanine, Glutamine, Threonine, Glycine, Tryptophan, Proline, Valine, Serine, Tyrosine, Asparagine, Selenocysteine, Pyrrolysine. In some embodiments, DDs of the present invention may be stabilized by ligands such as Sildenafil, Vardenafil, Tadalafil, Avanafil, Lodenafil, Mirodenafil, Udenafil, Benzamidenafil, Dasantafil, and Beminafil.

The amino acid sequences of the destabilizing domains encompassed in the invention have at least about 40%, 50 or 60% identity, further at least about 70% identity, preferably at least about 75% or 80% identity, more preferably at least about 85%, 86%, 87%, 88%, 89% or 90% identity, and further preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth therein. Percent identity may be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version Magic-BLAST 1.2.0, available from the National Institutes of Health. The BLAST program is based on the alignment method discussed in Karl and Altschul (1990) Proc. Natl. Acad. Sci USA, 87:2264-68 (the contents of which are incorporated by reference in their entirety).

Several hPDE5 destabilizing mutants were identified and are provided in Table 1. The position of the mutated amino acids listed in Table 1 is relative to the full length hPDE5 of SEQ ID NO. 1. The domains described in Table 1 include the catalytic domain of hPDE5 (e.g., 535-860 of SEQ ID NO. 1). In some embodiments, the hPDE5 DDs described in Table 1 may include a methionine at the N terminal of the catalytic domain of hPDE5, i.e. amino acid 535-860 of PDE5 WT. In Table 1, the mutated amino acids are in bold and underlined.

TABLE 1 hPDE5 DDs hPDE5 AA NA mutant SEQ ID SEQ ID description Amino Acid Sequence NO NO hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 5 350 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL W853F) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQK

QAL AEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 6 351 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, I821A) DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGF

DAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 7 352 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL Y829A) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQL

EALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 8 353 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, F787A) DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEF

DQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 9 354; (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 355 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, F736A) DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEF

ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQ QRIAELVAIEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 10 356 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, DL

HRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL D656L) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 11 357 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL Y728L) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLAL

IKRRGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 12 358; (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 359 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, R732L) DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKR

GEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 13 360 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQC

FAALKAGKIQNKLTDLEILALLIAALSHD WT, M625I) LDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILN SPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFFE LIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRI AELVAIEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQV GFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQALAE QQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 340 14 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 535-860 of SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR W853F) GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQK

QALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 341 15 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 535-860 of SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, I821A) MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVAIEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGF

DAICLQLYEALTHVSEDCFPLLDGCRKNRQK WQALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 342 16 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 535-860 of SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR Y829A) GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQL

EALTHVSEDCFPLLDGCRKNRQKW QALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 343 17 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 535-860 of SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, F787A) MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVATEF

DQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW QALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 344 18 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 535-860 of SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, F736A) MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR GEF

ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW QALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 345 19 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 535-860 of SHDL

HRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR D656L) GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW QALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 346 20 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 535-860 of SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLAL

IKRRG Y728L) EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 238 21 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 535-860 of SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, R732L) MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKR

GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW QALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 347 22 hPDE5 TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY (Amino acid HNWRHAFNTAQC

FAALKAGKIQNKLTDLEILALLIAALS 535-860 of HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMI WT, M625I) LNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEF FELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQ QRIAELVAIEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 348 361 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSA WT, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL H653A) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 349 362 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL D764A) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFF ELIRKNQFNLEDPHQKELFLAMLMTACALSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ

In some embodiments, DDs derived from hPDE5 may include one, two, three, four, five, or more of the mutations described in the previous Table (Table 1).

In some embodiments, DDs derived from hPDE5 may further comprise one, two, three, four, five or more mutations to the catalytic domain of hPDE5 and may be selected from E535D, E536G, Q541R, K555R, S560G, F559L, F561L, F564L, F564S, S766F, V585A, N587S, K591E, I599V, K604E, K608E, N609H, K630R, K633E, N636S, I648V, N661S, S663P, L675P, Y676D, Y676N, C677R, H678R, D687A, T711A, T712S, D724N, L738H, N742S, F744L, L746S, F755L, A762S, D764V, D764N, D764G, K795E, L797F, I799T, L804P, T802P, S815C, M816A, M816T, I824T, C839S, F840S, and K852E (as listed in Table 2). The position of the mutated amino acid is with respect to the hPDE5 of SEQ ID NO. 1. In Table 2, the mutated amino acids are underlined and in bold. The position of the mutated amino acids listed in Table 2 is relative to the full length hPDE5 of SEQ ID NO. 1

TABLE 2 Additional hPDE5 DDs hPDE5 mutant AA SEQ description Amino Acid Sequence ID NO hPDE5 (Amino

ETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 23 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, E535D) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino E

TRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT  24 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, E536G) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETREL

SLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 25 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, Q541R) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTL

ITDFSFSDFELSDLETALCTIRMFT 26 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, K555R) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITD

SFSDFELSDLETALCTIRMFT 27 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, F559L) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFS

SDFELSDLETALCTIRMFT 28 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, F561L) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSD

ELSDLETALCTIRMFT 29 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, F564L) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSD

ELSDLETALCTIRMFT 30 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, F564S) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 31 acid 535-860 of DLNLVQNFQM

HEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, K591E) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 32 acid 535-860 of DLNLVQ

FQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTA WT, N587S) QCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQ RSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT TLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFLA MLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEPT DLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGC RKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 33 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVK

NYRKNVAYHNWRHAFNT WT, K604E) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 34 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYR

NVAYHNWRHAFNT WT, K608E) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 35 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRK

VAYHNWRHAFNT WT, N609H) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 36 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, K630R) AQCMFAAL

AGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 37 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, K633E) AQCMFAALKAG

IQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 38 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, N636S) AQCMFAALKAGKIQ

KLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 39 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, N661S) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGV

NSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 40 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, Y676D) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQL

CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 41 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, Y676N) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQL

CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 42 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, C677R) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLY

HSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 43 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, H678R) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYC

SIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 44 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, D687A) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHF

QCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 45 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, T712S) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK T

LKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 46 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, D724N) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILAT

LALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 47 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, D724G) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILAT

LALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 48 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, L738H) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFE

IRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 49 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, N742S) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRK

QFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 50 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, A762S) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMT

CDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 51 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, D764N) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTAC

LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 52 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, D764G) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTAC

LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 53 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, D764V) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTAC

LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 54 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, S766F) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDL

AITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 55 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, K795E) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRER

ELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 56 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, L797F) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKE

NIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 57 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, I799T) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELN

E PTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLD GCRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 58 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, T802P) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP

DLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 59 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, S815C) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIP

MQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 60 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, M816A) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPS

QVGFIDAICLQLYEALTHVSEDCFPLLDGC RKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 61 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, I824T) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDA

CLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 62 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, C839S) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSED

FPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 63 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, K852E) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQ

WQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDF

FSDFELSDLETALCTIRMF 64 acid 535-860 of TDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, S560G) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 65 acid 535-860 of DLNL

QNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, V585A) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 66 acid 535-860 of DLNLVQNFQMKHEVLCRW

LSVKKNYRKNVAYHNWRHAFNT WT, I599V) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 67 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, I648V) AQCMFAALKAGKIQNKLTDLEILALL

AALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 68 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, S663P) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNN

YI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 69 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, L675P) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQ

YCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 70 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, T711A) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK

TLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 71 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, F744L) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQ

NLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 72 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, L746S) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFN

EDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 73 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, F755L) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKEL

L AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 74 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, L804P) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TD

MNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 75 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, M816T) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPS

QVGFIDAICLQLYEALTHVSEDCFPLLDGC RKNRQKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 76 acid 535-860 of DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT WT, F840S) AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDC

PLLDG CRKNRQKWQALAEQQ

In some embodiments, DDs derived from hPDE5 may include the combination of at least one, two, three, four or five mutations listed in Table 1 with at least 0, 1, 2, 3, 4, 5 or more mutations listed in Table 2.

In some embodiments, the DDs may be derived from hPDE5 (SEQ ID NO. 3), by mutating one or more amino acids residues between positions 530-550, 550-570, 570-590, 590-610, 620-640, 640-660, 660-680, 680-700, 710-730, 730-750, 750-770, 770-790, 790-810-830, 830-850, 850-860 of the catalytic domain of human PDE5 (SEQ ID NO. 1). In some embodiments, the mutation may be a conserved (with similar physicochemical properties as the amino acid at the mutation site), a semi conserved (e.g., negatively to positively charge amino acid) or a non-conserved (amino acid with different physicochemical properties than the amino acid at the mutation site). In some embodiments, the amino acid lysine may be mutated to glutamic acid or arginine; the amino acid phenylalanine may be mutated to leucine; the amino acid leucine may be mutated to phenylalanine; or the amino acid asparagine may be mutated to serine. Regions or portions or domains of wild type proteins may be utilized as SREs/DDs in whole or in part. They may be combined or rearranged to create new peptides, proteins, regions or domains of which any may be used as SREs/DDs or the starting point for the design of further SREs and/or DDs.

In some embodiments, DDs may be derived from hPDE5 by mutating amino acid residues conserved within all members of the PDE family. Exemplary conserved residues include but are not limited to, E682, H613, H617, H653, D654, and H657 residues of full length human PDE5 (SEQ ID NO. 1) and are taught in Sung et al. Nature (2003) 425, 98-102 (the contents of which are incorporated herein by reference in their entirety). In other embodiments, residues that are critical for binding to metals such as zinc and magnesium may be mutated to identify novel hPDE5 DDs. In some embodiments, hPDE5 derived DDs may be identified from a library of mutants of hPDE5 catalytic domain generated using a combination of error-prone PCR and nucleotide analog mutagenesis through random mutagenesis. Any of the mutations identified by site directed mutagenesis may be combined with the mutations identified by random mutagenesis. In some embodiments, DDs described herein may be derived by mutating the Y612 amino acid of hPDE5 (SEQ ID NO. 1). In some embodiments, the mutations to the Y612 amino acid may be combined with any of the mutations described herein. Independent co-crystals of hPDE5 with vardenafil and with sildenafil, have demonstrated that one of the rings of the both ligand, interacts with Y612 of hPDE5, an amino acid located within the catalytic site of hPDE5. Interactions occur via a hydrogen bond with a water molecule and via hydrophobic bonds. Y612F mutation, which ablates the hydrogen bonding potential, increases the inhibition of hPDE5 activity by both ligands. The Y612A mutation, which leads to the ablation of both hydrogen bonding and hydrophobic bonding potential has been shown to weaken the inhibition of hPDE5 catalytic activity by vardenafil and sildenafil to a lesser extent. These studies suggest that hydrophobic bonding involving Y612 is stronger for vardenafil than for sildenafil (Corbin et al. 2006, International Journal of Impotence Research 18, 251-257; the contents of which are incorporated by reference in their entirety).

The destabilization domains described herein may also include amino acid and nucleotide substitutions that do not affect stability, including conservative, non-conservative substitutions and or polymorphisms.

In some embodiments, hPDE5 DDs described herein may also be fragments of the above destabilizing domains, including fragments containing variant amino acid sequences. Preferred fragments are unstable in the absence of the stimulus and stabilized upon addition of the stimulus. Preferred fragments retain the ability to interact with the stimulus with similar efficiency as the DDs described herein.

In one embodiment, hPDE5 mutants are fused to AcGFP through a linker sequence at either the N-terminal or the C-terminal end of the fusion constructs. The AcGFP of Uniprot ID BAE93141 (SEQ ID NO. 363), may be used as the GFP template and is referred to as the wildtype or “WT” version of AcGFP. In some embodiments, the hPDE5 mutants described herein may also be operably linked to a luciferase (luc) gene, such as the firefly luciferase (Fluc) or Renilla luciferase (Rluc). The position of the mutations in the Fluc protein sequence described herein are based on the comparison of the SEQ ID NO. 223 with the wildtype luciferase sequence of Photinus pyralis (Uniprot ID: P08659.1) or “Fluc WT”, comprising the amino acid sequence of SEQ ID NO. 364. The destabilizing and ligand dependent stabilization properties of the fusion proteins may be evaluated by methods such as western blotting, and FACS. hPDE5 mutants that are fused to the N terminus of GFP are provided in Table 3. All constructs may be cloned into any vector known in the art and/or described herein such as, but not limited to, pLVX.IRES Puro vectors. OT-hPDE5C-036 (OT-001232) was placed under the transcriptional control of the EF1a promoter, while the other constructs described in Table 3 were placed under the transcriptional control of CMV promoter. In Table 3, and asterisk indicates the translation of the stop codon. Table 3 also provides alternate aliases for a given construct ID. These aliases are identified by the prefix OT.

TABLE 3 hPDE5-AcGFP constructs Construct AA NA ID/ SEQ ID SEQ ID Description Amino Acid Sequence NO. NO. Linker GGSGGGSGG  77  92 Linker GGSGGG  78  93; 300 Linker SG — AGT GGT AcGFP VSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYG  79 372 (Amino acid KLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQ 2-239 of WT) HDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLV NRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKN GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELYK AcGFP MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 365  94, (Amino acid GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 373 1-239 of WT) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K Fluc (Amino EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTI 366 374 acid 2-549 of AFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIV WT, N50D, VCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMG N119G, ISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGF S548I, QSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGST K549A, GLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFH L550V) HGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQS ALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGE AVAKRFHLPGIRQGYGLIETTSAILITPEGDDKPGAVGKV VPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNN PEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKY KGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAV VVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVP KGLTGKLDARKIREILIKAKKGGKIAV OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  80  95 001 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001075, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-001) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR hPDE5 GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW (Amino acid PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI 535-860 of PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW WT); linker QALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNG (GGSGGGSGG); HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL AcGFP SYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDG (Amino acid NYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEY 2-239 of NYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHY WT); stop QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFG FVTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  81  96 002 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001078, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-002) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR hPDE5 GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW (Amino acid PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI 535-860 of PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKF WT, W853F); QALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNG linker HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL (GGSGGGSGG); SYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDG AcGFP NYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEY (Amino acid NYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHY 2-239 of QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFG WT); stop) FVTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  82  97 003 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001080, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-003) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR hPDE5 GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW (Amino acid PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI 535-860 of PSMQVGFADAICLQLYEALTHVSEDCFPLLDGCRKNRQK WT, I821A); WQALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVN linker GHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTT (GGSGGGSGG); LSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD AcGFP GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKME (Amino acid YNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADH 2-239 of YQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF WT); stop GFVTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  83  98 004 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001081, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-004) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR hPDE5 GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW (Amino acid PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI 535-860 of PSMQVGFIDAICLQLAEALTHVSEDCFPLLDGCRKNRQKW WT, Y829A); QALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNG linker HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL (GGSGGGSGG); SYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDG AcGFP NYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEY (Amino acid NYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHY 2-239 of QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFG WT); stop FVTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  84  99 005 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001074, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-005) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR hPDE5 GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW (Amino acid PIQQRIAELVATEFADQGDRERKELNIEPTDLMNREKKNKI 535-860 of PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW WT, F787A); QALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNG linker HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL (GGSGGGSGG); SYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDG AcGFP NYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEY (Amino acid NYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHY 2-239 of QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFG WT); stop FVTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  85 100 006 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001076, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-006) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR hPDE5 GEFAELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW (Amino acid PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI 535-860 of PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW WT, F736A); QALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNG linker HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL (GGSGGGSGG); SYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDG AcGFP NYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEY (Amino acid NYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHY 2-239 of QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFG WT); stop FVTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  86 101 007 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001082, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-007) SHDLLHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR hPDE5 GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW (Amino acid PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI 535-860 of PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW WT, D656L); QALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNG linker HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL (GGSGGGSGG); SYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDG AcGFP NYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEY (Amino acid NYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHY 2-239 of QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFG WT); stop FVTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  87 102 008 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001083, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-008) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALLIKRRG hPDE5 EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI (Amino acid QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS 535-860 of MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ WT, Y728L); ALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNGH linker KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLS (GGSGGGSGG); YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGN AcGFP YKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYN (Amino acid YNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ 2-239 of QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGF WT); stop VTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  88 103 009 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001084, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-009) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG hPDE5 EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI (Amino acid QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS 535-860 of MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ WT, R732L); ALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNGH linker KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLS (GGSGGGSGG); YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGN AcGFP YKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYN (Amino acid YNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ 2-239 of QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGF WT); stop VTAAAITHGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  89 104 010 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001070, OT- HNWRHAFNTAQCIFAALKAGKIQNKLTDLEILALLIAALS hPDE5-010) HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMI Methionine; LNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEF hPDE5 FELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQ (Amino acid QRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS 535-860 of MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ WT, M625I); ALAEQQGGSGGGSGGVSKGAELFTGIVPILIELNGDVNGH linker KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLS (GGSGGGSGG); YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGN AcGFP YKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYN (Amino acid YNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ 2-239 of QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGF WT); stop VTAAAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC  90 105 028 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001224) HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL Methionine; SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL hPDE5 MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR (Amino acid GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW 535-860 of PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI WT); linker PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW (GGSGGG); QALAEQQGGSGGGVSKGAELFTGIVPILIELNGDVNGHKF AcGFP SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSYG (Amino acid VQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYK 2-239 of SRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYN WT); stop AHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVT AAAITHGMDELYK* OT-hPDE5C- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY  91 106 036 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001232; OT- QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL hPDE5-036) VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK AcGFP NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN (Amino acid HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY 1-239 of KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA WT); linker LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (SG); hPDE5 YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA (Amino acid LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL 535-860 of MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR WT); stop GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW QALAEQQ* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 367 375 031 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001227) HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL Methionine; SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL hPDE5 MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR (Amino acid GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW 535-860 of PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI WT); linker PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW (SG); Fluc QALAEQQSGEDAKNIKKGPAPFYPLEDGTAGEQLHKAMK (N50D, RYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYG N119G, LNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNE S548I, RELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMD K549A, SKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIA L550V); stop LIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPD TAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLR SLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGG APLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDD KPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGP MIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFI VDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPD DDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLR GGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 368 376 032 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001228) HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL Methionine; SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL hPDE5 MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR (Amino acid GEFAELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW 535-860 of PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI WT, F736A); PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW linker (SG); QALAEQQSGEDAKNIKKGPAPFYPLEDGTAGEQLHKAMK Fluc (N50D, RYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYG N119G, LNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNE S548I, RELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMD K549A, SKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIA L550V); stop LIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPD TAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLR SLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGG APLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDD KPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGP MIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFI VDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPD DDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLR GGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 369 377 033 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001229) HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL Methionine; SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL hPDE5 MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG (Amino acid EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI 535-860 of QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS WT, R732L); MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ Linker (SG); ALAEQQSGEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKR Fluc (N50D, YALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGL N119G, NTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNER S548I, ELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDS K549A, KTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALI L550V); stop MNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTA ILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSL QDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAP LSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKP GAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMI MSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVD RLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDD AGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGG VVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 370 378 086 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001211) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); hPDE5 LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSADLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, H653A); MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR stop GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW QALAEQQ* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 371 379 087 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001208) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); hPDE5 LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, D764A); MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR stop GEFFELIRKNQFNLEDPHQKELFLAMLMTACALSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW QALAEQQ*

In addition to human PDE5, other phosphodiesterases may also be used to generate novel destabilizing domains. The human PDE superfamily includes 11 structurally related but functionally distinct gene families (hPDE1 to hPDE11). These differ in their cellular functions, primary structures, affinities for cAMP and cGMP, catalytic properties and response to specific activators, inhibitors, effectors and mechanisms of regulation. As modular proteins, hPDEs exhibit a common structural organization with divergent amino-terminal regulatory regions and conserved carboxy-terminal catalytic core. The hPDE family proteins contain an N terminal regulatory region and a C terminal catalytic region. The N-terminal regulatory regions contain structural determinants that target individual hPDEs to different subcellular locations, and allow individual hPDEs to specifically respond to different post translational modification. The structural elements include dimerization domains, auto-inhibitory modules, binding sites for ligands and allosteric effectors. In contrast, the X ray crystal structure isolated catalytic domains of nine hPDE families (hPDE1 to hPDE5 and hPDE7 to hPDE10) have demonstrated that the catalytic domains of hPDEs share a similar topography, composed of ˜350 amino acids folded into 16 helices. Across the hPDE families, the active site forms a deep hydrophobic pocket that contains a hPDE specific, histidine-containing signature motif, HD(X₂) H(X₄) N (SEQ ID NO: 8377), and binding sites for two divalent metal ions that are essential for catalytic function. The affinity of hPDEs to specific cyclic nucleotides varies within the family-some hPDEs specifically hydrolyze cAMP (hPDE4, hPDE7 and hPDE8), whereas others hydrolyze cGMP (hPDE5, hPDE6 and hPDE9), and some hydrolyze both cAMP and cGMP (hPDE1, hPDE2, hPDE3, hPDE10 and hPDE11).

Similar to hPDE5, the catalytic domain or other functional domain of any hPDE family member may be mutagenized and screened for destabilizing mutations. Known inhibitors for each hPDE protein may also be tested for ligand-dependent stabilization.

In some embodiments, known mutations in phosphodiesterases that affect protein stability may be utilized to identify novel hPDE derived DDs. Mutations previously identified include, but are not limited to, hPDE5 (I778T), or hPDE6C (H602L), hPDE6C (E790K), hPDE6C (R104W), hPDE6C (Y323N), and hPDE6C (P391L) or hPDE4D (S752A), hPDE4D (S754A), hPDE4D (S752A, S754A), and hPDE4D (E757A, E758A, D759A) (Zhu et al. (2010) Mol Cell Biol. 4379-4390; Alexandre et al. (2015). Endocr. Relat. Cancer 22(4):519-30; Cheguru P. et al. (2015) Mol Cell Neurosci; 64: 1-8; the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, novel DDs may be generated from hPDE1 which is also known as calcium and calmodulin dependent phosphodiesterase. It has three subtypes hPDE1A, hPDE1B and hPDE1C. The enzyme contains three functional domains; a conserved catalytic core, a regulatory N-terminus and a C-terminus. The catalytic domains of hPDE1 have three helical subdomains; an N-terminal cyclin fold region, a linker region and a C-terminal helical bundle. Vinpocetine is a known inhibitor of hPDE1.

In some embodiments, novel DDs may be generated from hPDE2, a dual substrate enzyme that hydrolyzes both cAMP and cGMP. The distinguishing feature of this hPDE is that it is allosterically stimulated by cGMP binding to one of its GAF domains. The crystal structure of hPDE2 GAF-B domain reveals that the GAF-B domain binds cGMP with high affinity and selectivity. Exemplary hPDE2 inhibitors include EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), Oxindole, PDP and BAY 60-7550. Inhibitors that selectively inhibit a hPDE2 isoform may also be used, for example, substituted pyrido (2,3-b) pyrazines having a hPDE2A selective inhibitory action (See, e.g., U.S. Pat. No. 9,527,841; the contents of which are incorporated by reference in its entirety.)

In some embodiments, novel DDs may be generated from hPDE3 which preferentially hydrolyses cAMP. Like other hPDE family members, it comprises three functional domains, a conserved catalytic core, a regulatory terminus and the C-terminus. The catalytic core of hPDE is characterized by a unique 44-amino acid insert. Amrinone, Cilostazol, Milrinone, Enoximone and Pimobendan are inhibitors for hPDE3 enzyme.

In some embodiments, novel DDs may be generated from hPDE4, the principal second messenger for immune response regulation and is responsible for the hydrolysis of cAMP. Four isoforms of hPDE4 exist with each isoform having a unique N terminal region that specifies cellular localization by mediating interactions with scaffolding proteins which may further comprise upstream conserved regions (UCRs). All isoforms share invariant catalytic domain. The catalytic pocket is lined with highly conserved and invariant residues, including an invariant glutamine (Q369) that forms crucial hydrogen bond with substrates. Analyses of crystal structure of hPDE-inhibitor complexes suggest that two conserved residues are essential for inhibitor binding. The formation of hydrogen bonds with invariant glutamine determines the orientation of the inhibitors and conserved hydrophobic residues (1336 and F340) form a hydrophobic clamp that anchors inhibitors in the pocket. A number of small molecules can inhibit hPDE 4 activity, some of which are FDA approved such as AN2728 (4-[(1-hydroxy-1,3-dihydro-2,1-benzoxaborol-5-yl) oxy]benzonitrile), Apremilast/CC10004 (N-{2-[(1S)-1-(3-Ethoxy-4-methoxyphenyl)-2-(methyl sulfonyl) ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}acetamide), and Roflumilast. Other small molecules that inhibit hPDE4 also include E6005/RVT501, Cilomilast/SB-207,499, Ibudilast (AV-411 or MN-166), Mesembrenone, Piclamilast/RP 73401, Rolipram, Atizoram/CP-80633, Arofylline, CC-1088, Catramilast, CGH-2466, Cipamfylline, Drotaverine, Filaminast/WAY-PDA 641, HT-0712, DNS-001, ICI-63197, Indimilast, Irsogladine/MN 1695, Lirimilast/BAY 19-8004, Oglemilast, Revamilast, Ro 20-1724, Ronomilast, GSK256066, DC-TA-46, AWD 12-281 and YM-976.

In some embodiments, novel DDs may be generated from hPDE6. This holoenzyme includes hPDE6 alpha, hPDE6 beta and/or two identical inhibitory subunits of hPDE6 gamma. hPDE6 alpha and beta forms comprise three domains: two N-terminal GAF domains and one C-terminal catalytic domain. The non-catalytic GAF domains are responsible for cGMP binding.

In some embodiments, novel DDs may be generated from hPDE7, a cAMP specific hPDE which consists of two genes, hPDE7A and hPDE7B. There are no known regulatory domains on the N terminus as established for most of the other hPDE families, although consensus PKA phosphorylation sites exist in this region. Several small molecules can inhibit PDE7, including BRL-50481(N, N,2-Trimethyl-5-nitrobenzenesulfonamide) and ASB16165 (1H-Thieno(2,3-C) pyrazole-5-carboxamide, 1-cyclohexyl-N-(6-(4-hydroxy-1-piperidinyl)-3-pyridinyl)-3-methyl).

In some embodiments, novel DDs may be generated from hPDE8. Two subfamilies of hPDE8 exist and both they have very high affinity for the substrate cAMP and are insensitive to the non-specific PDE inhibitor IBMX. Each protein contains a catalytic core, a PAS (Per, Arnt and Sim) and a REC (receiver) domain. The crystal structure of the catalytic core of hPDE8 identified Tyr748 residue as a unique residue that distinguishes hPDE8 inhibitor binding from other hPDE proteins bound to inhibitors. PF-04957325 (Pfizer) is a small molecule inhibitor of hPDE8.

In some embodiments, novel DDs may be generated from hPDE9, which has the highest affinity for cGMP. The primary structure of hPDE9A is simple as it does not appear to contain any GAF domains or other N-terminal regulatory sequences found in other hPDEs. The catalytic pocket is lined with highly conserved and invariant residues, including an invariant glutamine Gln 453 in hPDE9A2 that forms crucial hydrogen bond with substrates. The formation of hydrogen bonds with invariant glutamine determines the orientation of the inhibitors and conserved hydrophobic residues form a hydrophobic clamp that anchors inhibitors in the pocket and wedges their ring structures against F456 in hPDE9A. Tested hPDE9 inhibitors may include BAY73-6691 (1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one), PF-04447943 (6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(oxan-4-yl)-2H-pyrazolo[3,4-d]pyrimidin-4-one) and WYQ-C28L.

In some embodiments, novel DDs may be generated from hPDE10, which can hydrolyze both cAMP and cGMP. Like some other hPDE family proteins, hPDE10 comprises 2 GAF domains in the N terminal region, a Protein Kinase A phosphorylation site and a catalytic domain. The catalytic pocket is lined with highly conserved and invariant residues, including an invariant glutamine Q726 in hPDE10A2 that forms crucial hydrogen bond with substrates. Several PDE10 inhibitors are under clinical trials including OMS 824, Papaverine and PF-2545920 (2-(4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl) phenoxymethyl) quinolone).

In some embodiments, novel DDs may be generated from hPDE11. Like hPDE10, the recently discovered hPDE11 can hydrolyze both cAMP and cGMP. It comprises of only one gene with four isoform variants. The longest variant, hPDE11A4, has two N-terminal GAF domains, whereas the other variants are truncations of this variant of varying lengths.

In some embodiments, any of the destabilizing mutations related to hPDE5 described herein may be structurally mapped onto other phosphodiesterases to generate destabilizing domains. In one embodiment, mutations that destabilize hPDE5, and which subsequently result in stabilization in the presence of sildenafil and/or vardenafil may be engineered onto hPDE6. In one embodiment, mutations that destabilize hPDE5, and which subsequently result in stabilization in the presence of Tadalafil may be engineered onto hPDE11.

The full length hPDEs and their catalytic domains that may be utilized to derive novel DDs are listed in Table 4.

TABLE 4 Sequences of human PDE proteins and their catalytic domains PDE protein (Uniprot ID or AA domain SEQ ID description) Amino Acid sequence NO. hPDE1A MDDHVTIRKKHLQRPIFRLRCLVKQLERGDVNVVDLKKNIEYAA 107 (Uniprot ID: SVLEAVYIDETRRLLDTEDELSDIQTDSVPSEVRDWLASTFTRKM P54750) GMTKKKPEEKPKFRSIVHAVQAGIFVERMYRKTYHMVGLAYPA AVIVTLKDVDKWSFDVFALNEASGEHSLKFMIYELFTRYDLINRF KIPVSCLITFAEALEVGYSKYKNPYHNLIHAADVTQTVHYIMLHT GIMHWLTELEILAMVFAAAIHDYEHTGTTNNFHIQTRSDVAILYN DRSVLENHHVSAAYRLMQEEEMNILINLSKDDWRDLRNLVIEMV LSTDMSGHFQQIKNIRNSLQQPEGIDRAKTMSLILHAADISHPAKS WKLHYRWTMALMEEFFLQGDKEAELGLPFSPLCDRKSTMVAQS QIGFIDFIVEPTFSLLTDSTEKIVIPLIEEASKAETSSYVASSSTTIVG LHIADALRRSNTKGSMSDGSYSPDYSLAAVDLKSFKNNLVDIIQQ NKERWKELAAQEARTSSQKCEFIHQ hPDE1A FKIPVSCLITFAEALEVGYSKYKNPYHNLIHAADVTQTVHYIMLH 108 Catalytic TGIMHWLTELEILAMVFAAAIHDYEHTGTTNNFHIQTRSDVAILY domain (Amino NDRSVLENHHVSAAYRLMQEEEMNILINLSKDDWRDLRNLVIEM acid 193-515 VLSTDMSGHFQQIKNIRNSLQQPEGIDRAKTMSLILHAADISHPAK of PDE1A) SWKLHYRWTMALMEEFFLQGDKEAELGLPFSPLCDRKSTMVAQ SQIGFIDFIVEPTFSLLTDSTEKIVIPLIEEASKAETSSYVASSSTTIVG LHIADALRRSNTKGSMSDGSYSPDYSLAAVDLKSFKNNLVDIIQQ NKERW hPDE1B MELSPRSPPEMLEESDCPSPLELKSAPSKKMWIKLRSLLRYMVKQ 109 (Uniprot ID: LENGEINIEELKKNLEYTASLLEAVYIDETRQILDIEDELQELRSD Q01064) AVPSEVRDWLASTFTQQARAKGRRAEEKPKFRSIVHAVQAGIFV ERMFRRTYTSVGPTYSTAVLNCLKNLDLWCFDVFSLNQAADDH ALRTIVFELLTRHNLISRFKIPTVFLMSFLDALETGYGKYKNPYHN QIHAADVTQTVHCFLLRTGMVHCLSEIELLAIIFAAAIHDYEHTGT TNSFHIQTKSECAIVYNDRSVLENHHISSVFRLMQDDEMNIFINLT KDEFVELRALVIEMVLATDMSCHFQQVKTMKTALQQLERIDKPK ALSLLLHAADISHPTKQWLVHSRWTKALMEEFFRQGDKEAELGL PFSPLCDRTSTLVAQSQIGFIDFIVEPTFSVLTDVAEKSVQPLADED SKSKNQPSFQWRQPSLDVEVGDPNPDVVSFRSTWVKRIQENKQK WKERAASGITNQMSIDELSPCEEEAPPSPAEDEHNQNGNLD hPDE1B FKIPTVFLMSFLDALETGYGKYKNPYHNQIHAADVTQTVHCFLLR 110 Catalytic TGMVHCLSEIELLAIIFAAAIHDYEHTGTTNSFHIQTKSECAIVYND domain (Amino RSVLENHHISSVFRLMQDDEMNIFINLTKDEFVELRALVIEMVLA acid 197-496 TDMSCHFQQVKTMKTALQQLERIDKPKALSLLLHAADISHPTKQ of PDE1B) WLVHSRWTKALMEEFFRQGDKEAELGLPFSPLCDRTSTLVAQSQ IGFIDFIVEPTFSVLTDVAEKSVQPLADEDSKSKNQPSFQWRQPSL DVEVGDPNPDVVSFRSTWVKRIQENKQKW hPDE1C MESPTKEIEEFESNSLKYLQPEQIEKIWLRLRGLRKYKKTSQRLRS 111 (Uniprot ID: LVKQLERGEASVVDLKKNLEYAATVLESVYIDETRRLLDTEDELS Q14123) DIQSDAVPSEVRDWLASTFTRQMGMMLRRSDEKPRFKSIVHAVQ AGIFVERMYRRTSNMVGLSYPPAVIEALKDVDKWSFDVFSLNEA SGDHALKFIFYELLTRYDLISRFKIPISALVSFVEALEVGYSKHKNP YHNLMHAADVTQTVHYLLYKTGVANWLTELEIFAIIFSAAIHDYE HTGTTNNFHIQTRSDPAILYNDRSVLENHHLSAAYRLLQDDEEM NILINLSKDDWREFRTLVIEMVMATDMSCHFQQIKAMKTALQQP EAIEKPKALSLMLHTADISHPAKAWDLHHRWTMSLLEEFFRQGD REAELGLPFSPLCDRKSTMVAQSQVGFIDFIVEPTFTVLTDMTEKI VSPLIDETSQTGGTGQRRSSLNSISSSDAKRSGVKTSGSEGSAPINN SVISVDYKSFKATWIEVVHINRERWRAKVPKEEKAKKEAEEKAR LAAEEQQKEMEAKSQAEEGASGKAEKKTSGETKNQVNGTRANK SDNPRGKNSKAEKSSGEQQQNGDFKDGKNKTDKKDHSNIGNDS KKTDGTKQRSHGSPAPSTSSTCRLTLPVIKPPLRHFKRPAYASSSY APSVSKKTDEHPARYKMLDQRIKMKKIQNISHNWNRK hPDE1C FKIPISALVSFVEALEVGYSKHKNPYHNLMHAADVTQTVHYLLY 112 Catalytic KTGVANWLTELEIFAIIFSAAIHDYEHTGTTNNFHIQTRSDPAILYN domain (Amino DRSVLENHHLSAAYRLLQDDEEMNILINLSKDDWREFRTLVIEM acid 202-521 VMATDMSCHFQQIKAMKTALQQPEAIEKPKALSLMLHTADISHP of PDE1C) AKAWDLHHRWTMSLLEEFFRQGDREAELGLPFSPLCDRKSTMV AQSQVGFIDFIVEPTFTVLTDMIEKIVSPLIDETSQTGGTGQRRSSL NSISSSDAKRSGVKTSGSEGSAPINNSVISVDYKSFKATWTEVVHI NRERW hPDE2A MGQACGHSILCRSQQYPAARPAEPRGQQVFLKPDEPPPPPQPCAD 113 (Uniprot ID: SLQDALLSLGSVIDISGLQRAVKEALSAVLPRVETVYTYLLDGES 000408) QLVCEDPPHELPQEGKVREAIISQKRLGCNGLGFSDLPGKPLARL VAPLAPDTQVLVMPLADKEAGAVAAVILVHCGQLSDNEEWSLQ AVEKHTLVALRRVQVLQQRGPREAPRAVQNPPEGTAEDQKGGA AYTDRDRKILQLCGELYDLDASSLQLKVLQYLQQETRASRCCLL LVSEDNLQLSCKVIGDKVLGEEVSFPLTGCLGQVVEDKKSIQLKD LTSEDVQQLQSMLGCELQAMLCVPVISRATDQVVALACAFNKLE GDLFTDEDEHVIQHCFHYTSTVLTSTLAFQKEQKLKCECQALLQV AKNLFTHLDDVSVLLQEIITEARNLSNAEICSVFLLDQNELVAKVF DGGVVDDESYEIRIPADQGIAGHVATTGQILNIPDAYAHPLFYRG VDDSTGFRTRNILCFPIKNENQEVIGVAELVNKINGPWFSKFDEDL ATAFSIYCGISIAHSLLYKKVNEAQYRSHLANEMNIMYHMKVSDD EYTKLLHDGIQPVAAIDSNFASFTYTPRSLPEDDTSMAILSMLQD MNFINNYKIDCPTLARFCLMVKKGYRDPPYHNWMHAFSVSHFC YLLYKNLELTNYLEDIEIFALFISCMCHDLDHRGTNNSFQVASKS VLAALYSSEGSVMERHHFAQAIAILNTHGCNIFDHFSRKDYQRML DLMRDIILATDLAHHLRIFKDLQKMAEVGYDRNNKQHHRLLLCL LMTSCDLSDQTKGWKTTRKIAELIYKEFFSQGDLEKAMGNRPME MMDREKAYIPELQISFMEHIAMPIYKLLQDLFPKAAELYERVASN REHWTKVSHKFTIRGLPSNNSLDFLDEEYEVPDLDGTRAPINGCC SLDAE hPDE2A KIDCPTLARFCLMVKKGYRDPPYHNWMHAFSVSHFCYLLYKNL 114 Catalytic ELTNYLEDIEIFALFISCMCHDLDHRGTNNSFQVASKSVLAALYSS domain (Amino EGSVMERHHFAQAIAILNTHGCNIFDHFSRKDYQRMLDLMRDIIL acid 633-891 ATDLAHHLRIFKDLQKMAEVGYDRNNKQHHRLLLCLLMTSCDL of PDE2A) SDQTKGWKTTRKIAELIYKEFFSQGDLEKAMGNRPMEMMDREK AYIPELQISFMEHIAMPIYKLLQDLFPKAAELYERVASN hPDE3A MAVPGDAARVRDKPVHSGVSQAPTAGRDCHHRADPASPRDSGC 115 (Uniprot ID: RGCWGDLVLQPLRSSRKLSSALCAGSLSFLLALLVRLVRGEVGC Q14432) DLEQCKEAAAAEEEEAAPGAEGGVFPGPRGGAPGGGARLSPWL QPSALLFSLLCAFFWMGLYLLRAGVRLPLAVALLAACCGGEALV QIGLGVGEDHLLSLPAAGVVLSCLAAATWLVLRLRLGVLMIALT SAVRTVSLISLERFKVAWRPYLAYLAGVLGILLARYVEQILPQSA EAAPREHLGSQLIAGTKEDIPVFKRRRRSSSVVSAEMSGCSSKSHR RTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIAVMGEAH GLITDLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPVTS LSENYTCSDSEESSEKDKLAIPKRLRRSLPPGLLRRVSSTWTTTTS ATGLPTLEPAPVRRDRSTSIKLQEAPSSSPDSWNNPVMMTLTKSR SFTSSYAISAANHVKAKKQSRPGALAKISPLSSPCSSPLQGTPASSL VSKISAVQFPESADTTAKQSLGSHRALTYTQSAPDLSPQILTPPVIC SSCGRPYSQGNPADEPLERSGVATRTPSRTDDTAQVTSDYETNNN SDSSDIVQNEDETECLREPLRKASACSTYAPETMMFLDKPILAPEP LVMDNLDSIMEQLNTWNFPIFDLVENIGRKCGRILSQVSYRLFED MGLFEAFKIPIREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWY LTTQPIPGLSTVINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTD DKYGCLSGNIPALELMALYVAAAMHDYDHPGRTNAFLVATSAP QAVLYNDRSVLENHHAAAAWNLFMSRPEYNFLINLDHVEFKHF RFLVIEAILATDLKKHFDFVAKFNGKVNDDVGIDWTNENDRLLV CQMCIKLADINGPAKCKELHLQWTDGIVNEFYEQGDEEASLGLPI SPFMDRSAPQLANLQESFISHIVGPLCNSYDSAGLMPGKWVEDSD ESGDTDDPEEEEEEAPAPNEEETCENNESPKKKTFKRRKIYCQITQ HLLQNHKMWKKVIEEEQRLAGIENQSLDQTPQSHSSEQIQAIKEE EEEKGKPRGEEIPTQKPDQ hPDE3A FKIPIREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIP 116 Catalytic GLSTVINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGCL domain (Amino SGNIPALELMALYVAAAMHDYDHPGRTNAFLVATSAPQAVLYN acid 728-1086 DRSVLENHHAAAAWNLFMSRPEYNFLINLDHVEFKHFRFLVIEAI of PDE3A) LATDLKKHFDFVAKFNGKVNDDVGIDWTNENDRLLVCQMCIKL ADINGPAKCKELHLQWTDGIVNEFYEQGDEEASLGLPISPFMDRS APQLANLQESFISHIVGPLCNSYDSAGLMPGKWVEDSDESGDTDD PEEEEEEAPAPNEEETCENNESPKKKTFKRRKIYCQITQHLLQNHK MW hPDE3B MRRDERDAKAMRSLQPPDGAGSPPESLRNGYVKSCVSPLRQDPP 117 (Uniprot ID: RGFFFHLCRFCNVELRPPPASPQQPRRCSPFCRARLSLGALAAFVL Q13370) ALLLGAEPESWAAGAAWLRTLLSVCSHSLSPLFSIACAFFFLTCFL TRTKRGPGPGRSCGSWWLLALPACCYLGDFLVVVQWWSWPWGD GDAGSAAPHTPPEAAAGRLLLVLSCVGLLLTLAHPLRLRHCVLV LLLASFVVVWVSFTSLGSLPSALRPLLSGLVGGAGCLLALGLDHFF QIREAPLHPRLSSAAEEKVPVIRPRRRSSCVSLGETAASYYGSCKIF RRPSLPCISREQMILWDWDLKQWYKPHYQNSGGGNGVDLSVLN EARNMVSDLLTDPSLPPQVISSLRSISSLMGAFSGSCRPKINPLTPF PGFYPCSEIEDPAEKGDRKLNKGLNRNSLPTPQLRRSSGTSGLLPV EQSSRWDRNNGKRPHQEFGISSQGCYLNGPFNSNLLTIPKQRSSS VSLTHHVGLRRAGVLSSLSPVNSSNHGPVSTGSLTNRSPIEFPDTA DFLNKPSVILQRSLGNAPNTPDFYQQLRNSDSNLCNSCGHQMLK YVSTSESDGTDCCSGKSGEEENIFSKESFKLMETQQEEETEKKDSR KLFQEGDKWLTEEAQSEQQTNIEQEVSLDLILVEEYDSLIEKMSN WNFPIFELVEKMGEKSGRILSQVMYTLFQDTGLLEIFKIPTQQFMN YFRALENGYRDIPYHNRIHATDVLHAVVVYLTTRPVPGLQQIHNG CGTGNETDSDGRINHGRIAYISSKSCSNPDESYGCLSSNIPALELM ALYVAAAMHDYDHPGRTNAFLVATNAPQAVLYNDRSVLENHH AASAWNLYLSRPEYNFLLHLDHVEFKRFRFLVIEAILATDLKKHF DFLAEFNAKANDVNSNGIEWSNENDRLLVCQVCIKLADINGPAK VRDLHLKWTEGIVNEFYEQGDEEANLGLPISPFMDRSSPQLAKLQ ESFITHIVGPLCNSYDAAGLLPGQWLEAEEDNDIESGDDEDGEEL DTEDEEMENNLNPKPPRRKSRRRIFCQLMHHLIENHKIWKEIVEE EEKCKADGNKLQVENSSLPQADEIQVIEEADEEE hPDE3B FKIPTQQFMNYFRALENGYRDIPYHNRIHATDVLHAVVVYLTTRP 118 Catalytic VPGLQQIHNGCGTGNETDSDGRINHGRIAYISSKSCSNPDESYGCL domain (Amino SSNIPALELMALYVAAAMHDYDHPGRTNAFLVATNAPQAVLYN acid 713-1072 DRSVLENHHAASAWNLYLSRPEYNFLLHLDHVEFKRFRFLVIEAI of PDE3B) LATDLKKHFDFLAEFNAKANDVNSNGIEWSNENDRLLVCQVCIK LADINGPAKVRDLHLKWTEGIVNEFYEQGDEEANLGLPISPFMDR SSPQLAKLQESFITHIVGPLCNSYDAAGLLPGQWLEAEEDNDILS GDDEDGEELDTEDEEMENNLNPKPPRRKSRRRIFCQLMHHLTEN HKIW hPDE4A MEPPTVPSERSLSLSLPGPREGQATLKPPPQHLWRQPRTPIRIQQR 119 (Uniprot ID: GYSDSAERAERERQPHRPIERADAMDTSDRPGLRTTRMSWPSSFH P27815) GTGTGSGGAGGGSSRRFEAENGPTPSPGRSPLDSQASPGLVLHAG AATSQRRESFLYRSDSDYDMSPKTMSRNSSVTSEAHAEDLIVTPF AQVLASLRSVRSNFSLLTNVPVPSNKRSPLGGPTPVCKATLSEETC QQLARETLEELDWCLEQLETMQTYRSVSEMASHKFKRMLNREL THLSEMSRSGNQVSEYISTTFLDKQNEVEIPSPTMKEREKQQAPRP RPSQPPPPPVPHLQPMSQITGLKKLMHSNSLNNSNIPRFGVKTDQE ELLAQELENLNKWGLNIFCVSDYAGGRSLTCIMYMIFQERDLLK KFRIPVDTMVTYMLTLEDHYHADVAYHNSLHAADVLQSTHVLL ATPALDAVFTDLEILAALFAAAIHDVDHPGVSNQFLINTNSELAL MYNDESVLENHHLAVGFKLLQEDNCDIFQNLSKRQRQSLRKMVI DMVLATDMSKHMTLLADLKTMVETKKVTSSGVLLLDNYSDRIQ VLRNMVHCADLSNPTKPLELYRQWTDRIMAEFFQQGDRERERG MEISPMCDKHTASVEKSQVGFIDYIVHPLWETWADLVHPDAQEIL DTLEDNRDWYYSAIRQSPSPPPEEESRGPGHPPLPDKFQFELTLEE EEEEEISMAQIPCTAQEALTAQGLSGVEEALDATIAWEASPAQESL EVMAQEASLEAELEAVYLTQQAQSTGSAPVAPDEFSSREEFVVA VSHSSPSALALQSPLLPAWRTLSVSEHAPGLPGLPSTAAEVEAQR EHQAAKRACSACAGTFGEDTSALPAPGGGGSGGDPT hPDE4A QPMSQITGLKKLMHSNSLNNSNIPRFGVKTDQEELLAQELENLNK 120 Catalytic WGLNIFCVSDYAGGRSLTCIMYMIFQERDLLKKFRIPVDTMVTY domain (Amino MLTLEDHYHADVAYHNSLHAADVLQSTHVLLATPALDAVFTDL acid 330-723 of EILAALFAAAIHDVDHPGVSNQFLINTNSELALMYNDESVLENHH PDE4A) LAVGFKLLQEDNCDIFQNLSKRQRQSLRKMVIDMVLATDMSKH MTLLADLKTMVETKKVTSSGVLLLDNYSDRIQVLRNMVHCADL SNPTKPLELYRQWTDRIMAEFFQQGDRERERGMEISPMCDKHTA SVEKSQVGFIDYIVHPLWETWADLVHPDAQEILDTLEDNRDWYY SAIRQSPSPPPEEESRGPGHPPLPDKFQFELTLEEEEEEEISM hPDE4B MKKSRSVMTVMADDNVKDYFECSLSKSYSSSSNTLGIDLWRGRR 121 (Uniprot ID: CCSGNLQLPPLSQRQSERARTPEGDGISRPTTLPLTTLPSIAITTVSQ Q07343) ECFDVENGPSPGRSPLDPQASSSAGLVLHATFPGHSQRRESFLYRS DSDYDLSPKAMSRNSSLPSEQHGDDLIVTPFAQVLASLRSVRNNF TILTNLHGTSNKRSPAASQPPVSRVNPQEESYQKLAMETLEELDW CLDQLETIQTYRSVSEMASNKFKRMLNRELTHLSEMSRSGNQVS EYISNTFLDKQNDVEIPSPTQKDREKKKKQQLMTQISGVKKLMHS SSLNNTSISRFGVNTENEDHLAKELEDLNKWGLNIFNVAGYSHNR PLTCIMYAIFQERDLLKTFRISSDTFITYMMTLEDHYHSDVAYHNS LHAADVAQSTHVLLSTPALDAVFTDLEILAAIFAAAIHDVDHPGV SNQFLINTNSELALMYNDESVLENHHLAVGFKLLQEEHCDIFMNL TKKQRQTLRKMVIDMVLATDMSKHMSLLADLKTMVETKKVTSS GVLLLDNYTDRIQVLRNMVHCADLSNPTKSLELYRQWTDRIMEE FFQQGDKERERGMEISPMCDKHTASVEKSQVGFIDYIVHPLWET WADLVQPDAQDILDTLEDNRNWYQSMIPQSPSPPLDEQNRDCQG LMEKFQFELTLDEEDSEGPEKEGEGHSYFSSTKTLCVIDPENRDSL GETDIDIATEDKSPVDT hPDE4B VNTENEDHLAKELEDLNKWGLNIFNVAGYSHNRPLTCIMYAIFQ 122 Catalytic ERDLLKTFRISSDTFITYMMTLEDHYHSDVAYHNSLHAADVAQS domain (Amino THVLLSTPALDAVFTDLEILAAIFAAAIHDVDHPGVSNQFLINTNS acid 330-682 of ELALMYNDESVLENHHLAVGFKLLQEEHCDIFMNLTKKQRQTLR PDE4B) KMVIDMVLATDMSKHMSLLADLKTMVETKKVTSSGVLLLDNYT DRIQVLRNMVHCADLSNPTKSLELYRQWTDRIMEEFFQQGDKER ERGMEISPMCDKHTASVEKSQVGFIDYIVHPLWETWADLVQPDA QDILDTLEDNRNWYQSMIPQSPSPPLDEQNRDCQGLMEKFQFEL hPDE4C MENLGVGEGAEACSRLSRSRGRHSMTRAPKHLWRQPRRPIRIQQ 123 (Uniprot ID: RFYSDPDKSAGCRERDLSPRPELRKSRLSWPVSSCRRFDLENGLS Q08493) CGRRALDPQSSPGLGRIMQAPVPHSQRRESFLYRSDSDYELSPKA MSRNSSVASDLHGEDMIVTPFAQVLASLRTVRSNVAALARQQCL GAAKQGPVGNPSSSNQLPPAEDTGQKLALETLDELDWCLDQLET LQTRHSVGEMASNKFKRILNRELTHLSETSRSGNQVSEYISRTFLD QQTEVELPKVTAEEAPQPMSRISGLHGLCHSASLSSATVPRFGVQ TDQEEQLAKELEDTNKWGLDVFKVAELSGNRPLTAIIFSIFQERDL LKTFQIPADTLATYLLMLEGHYHANVAYHNSLHAADVAQSTHV LLATPALEAVFTDLEILAALFASAIHDVDHPGVSNQFLINTNSELA LMYNDASVLENHHLAVGFKLLQAENCDIFQNLSAKQRLSLRRM VIDMVLATDMSKHMNLLADLKTMVETKKVTSLGVLLLDNYSDR IQVLQNLVHCADLSNPTKPLPLYRQWTDRIMAEFFQQGDRERES GLDISPMCDKHTASVEKSQVGFIDYIAHPLWETWADLVHPDAQD LLDTLEDNREWYQSKIPRSPSDLTNPERDGPDRFQFELTLEEAEEE DEEEEEEGEETALAKEALELPDTELLSPEAGPDPGDLPLDNQRT hPDE4C VQTDQEEQLAKELEDTNKWGLDVFKVAELSGNRPLTAIIFSIFQE 124 Catalytic RDLLKTFQIPADTLATYLLMLEGHYHANVAYHNSLHAADVAQST domain (Amino HVLLATPALEAVFTDLEILAALFASAIHDVDHPGVSNQFLINTNSE acid 312-677 of LALMYNDASVLENHHLAVGFKLLQAENCDIFQNLSAKQRLSLRR PDE4C) MVIDMVLATDMSKHMNLLADLKTMVETKKVTSLGVLLLDNYS DRIQVLQNLVHCADLSNPTKPLPLYRQWTDRIMAEFFQQGDRER ESGLDISPMCDKHTASVEKSQVGFIDYIAHPLWETWADLVHPDA QDLLDTLEDNREWYQSKIPRSPSDLTNPERDGPDRFQFELTLEEA EEEDEEEEEEGE hPDE4D MEAEGSSAPARAGSGEGSDSAGGATLKAPKHLWRHEQHHQYPL 125 (Uniprot ID: RQPQFRLLHPHHHLPPPPPPSPQPQPQCPLQPPPPPPLPPPPPPPGAA Q08499) RGRYASSGATGRVRHRGYSDTERYLYCRAMDRTSYAVETGHRP GLKKSRMSWPSSFQGLRRFDVDNGTSAGRSPLDPMTSPGSGLILQ ANFVHSQRRESFLYRSDSDYDLSPKSMSRNSSIASDIHGDDLIVTP FAQVLASLRTVRNNFAALTNLQDRAPSKRSPMCNQPSINKATITE EAYQKLASETLEELDWCLDQLETLQTRHSVSEMASNKFKRMLNR ELTHLSEMSRSGNQVSEFISNTFLDKQHEVEIPSPTQKEKEKKKRP MSQISGVKKLMHSSSLTNSSIPRFGVKTEQEDVLAKELEDVNKW GLHVFRIAELSGNRPLTVIMHTIFQERDLLKTFKIPVDTLITYLMTL EDHYHADVAYHNNIHAADVVQSTHVLLSTPALEAVFTDLEILAAI FASAIHDVDHPGVSNQFLINTNSELALMYNDSSVLENHHLAVGFK LLQEENCDIFQNLTKKQRQSLRKMVIDIVLATDMSKHMNLLADL KTMVETKKVTSSGVLLLDNYSDRIQVLQNMVHCADLSNPTKPLQ LYRQWTDRIMEEFFRQGDRERERGMEISPMCDKHNASVEKSQVG FIDYIVHPLWETWADLVHPDAQDILDTLEDNREWYQSTIPQSPSP APDDPEEGRQGQTEKFQFELTLEEDGESDTEKDSGSQVEEDTSCS DSKTLCTQDSESTEIPLDEQVEEEAVGEEEESQPEACVIDDRSPDT hPDE4D VKTEQEDVLAKELEDVNKWGLHVFRIAELSGNRPLTVIMHTIFQE 126 Catalytic RDLLKTFKIPVDTLITYLMTLEDHYHADVAYHNNIHAADVVQST domain (Amino HVLLSTPALEAVFTDLEILAAIFASAIHDVDHPGVSNQFLINTNSEL acid 386-751 of ALMYNDSSVLENHHLAVGFKLLQEENCDIFQNLTKKQRQSLRKM PDE4D) VIDIVLATDMSKHMNLLADLKTMVETKKVTSSGVLLLDNYSDRI QVLQNMVHCADLSNPTKPLQLYRQWTDRIMEEFFRQGDRERER GMEISPMCDKHNASVEKSQVGFIDYIVHPLWETWADLVHPDAQD ILDTLEDNREWYQSTIPQSPSPAPDDPEEGRQGQTEKFQFELTLEE DGESDTEKD hPDE6A MGEVTAEEVEKFLDSNIGFAKQYYNLHYRAKLISDLLGAKEAAV 127 (Uniprot ID: DFSNYHSPSSMEESEIIFDLLRDFQENLQTEKCIFNVMKKLCFLLQ P16499) ADRMSLFMYRTRNGIAELATRLFNVHKDAVLEDCLVMPDQEIVF PLDMGIVGHVAHSKKIANVPNTEEDEHFCDFVDILTEYKTKNILA SPIMNGKDVVAIIMAVNKVDGSHFTKRDEEILLKYLNFANLIMKV YHLSYLHNCETRRGQILLWSGSKVFEELTDIERQFHKALYTVRAF LNCDRYSVGLLDMTKQKEFFDVWPVLMGEVPPYSGPRTPDGREI NFYKVIDYILHGKEDIKVIPNPPPDHWALVSGLPAYVAQNGLICNI MNAPAEDFFAFQKEPLDESGWMIKNVLSMPIVNKKEEIVGVATF YNRKDGKPFDEMDETLMESLTQFLGWSVLNPDTYESMNKLENR KDIFQDIVKYHVKCDNEEIQKILKTREVYGKEPWECEEEELAEILQ AELPDADKYEINKFHFSDLPLTELELVKCGIQMYYELKVVDKFHI PQEALVRFMYSLSKGYRKITYHNWRHGFNVGQTMFSLLVTGKL KRYFTDLEALAMVTAAFCHDIDHRGTNNLYQMKSQNPLAKLHG SSILERHHLEFGKTLLRDESLNIFQNLNRRQHEHAIHMMDIAIIAT DLALYFKKRTMFQKIVDQSKTYESEQEWTQYMMLEQTRKEIVM AMMMTACDLSAITKPWEVQSQVALLVAAEFWEQGDLERTVLQQ NPIPMMDRNKADELPKLQVGFIDFVCTFVYKEFSRFHEEITPMLD GITNNRKEWKALADEYDAKMKVQEEKKQKQQSAKSAAAGNQP GGNPSPGGATTSKSCCIQ hPDE6A EEEELAEILQAELPDADKYEINKFHFSDLPLTELELVKCGIQMYYE 128 Catalytic LKVVDKFHIPQEALVRFMYSLSKGYRKITYHNWRHGFNVGQTM domain (Amino FSLLVTGKLKRYFTDLEALAMVTAAFCHDIDHRGTNNLYQMKSQ acid 483-819 of NPLAKLHGSSILERHHLEFGKTLLRDESLNIFQNLNRRQHEHAIH PDE6A) MMDIAIIATDLALYFKKRTMFQKIVDQSKTYESEQEWTQYMMLE QTRKEIVMAMMMTACDLSAITKPWEVQSQVALLVAAEFWEQGD LERTVLQQNPIPMMDRNKADELPKLQVGFIDFVCTFVYKEFSRFH EEITPMLDGITNNRKEWKALADEYDAK hPDE6B MSLSEEQARSFLDQNPDFARQYFGKKLSPENVAAACEDGCPPDC 129 (Uniprot ID: DSLRDLCQVEESTALLELVQDMQESINMERVVFKVLRRLCTLLQ P35913) ADRCSLFMYRQRNGVAELATRLFSVQPDSVLEDCLVPPDSEIVFP LDIGVVGHVAQTKKMVNVEDVAECPHFSSFADELTDYKTKNML ATPIMNGKDVVAVIMAVNKLNGPFFTSEDEDVFLKYLNFATLYL KIYHLSYLHNCETRRGQVLLWSANKVFEELTDIERQFHKAFYTVR AYLNCERYSVGLLDMTKEKEFFDVWSVLMGESQPYSGPRTPDGR EIVFYKVIDYVLHGKEEIKVIPTPSADHWALASGLPSYVAESGFIC NIMNASADEMFKFQEGALDDSGWLIKNVLSMPIVNKKEEIVGVA TFYNRKDGKPFDEQDEVLMESLTQFLGWSVMNTDTYDKMNKLE NRKDIAQDMVLYHVKCDRDEIQLILPTRARLGKEPADCDEDELG EILKEELPGPTTFDIYEFHFSDLECTELDLVKCGIQMYYELGVVRK FQIPQEVLVRFLFSISKGYRRITYHNWRHGFNVAQTMFTLLMTGK LKSYYTDLEAFAMVTAGLCHDIDHRGTNNLYQMKSQNPLAKLH GSSILERHHLEFGKFLLSEETLNIYQNLNRRQHEHVIHLMDIAIIAT DLALYFKKRAMFQKIVDESKNYQDKKSWVEYLSLETTRKEIVMA MMMTACDLSAITKPWEVQSKVALLVAAEFWEQGDLERTVLDQQ PIPMMDRNKAAELPKLQVGFIDFVCTFVYKEFSRFHEEILPMFDRL QNNRKEWKALADEYEAKVKALEEKEEEERVAAKKVGTEICNGG PAPKSSTCCIL hPDE6B PADCDEDELGEILKEELPGPTTFDIYEFHFSDLECTELDLVKCGIQ 130 Catalytic MYYELGVVRKFQIPQEVLVRFLFSISKGYRRITYHNWRHGFNVA domain (Amino QTMFTLLMTGKLKSYYTDLEAFAMVTAGLCHDIDHRGTNNLYQ acid 476-817 of MKSQNPLAKLHGSSILERHHLEFGKFLLSEETLNIYQNLNRRQHE PDE6B) HVIHLMDIAIIATDLALYFKKRAMFQKIVDESKNYQDKKSWVEY LSLETTRKEIVMAMMMTACDLSAITKPWEVQSKVALLVAAEFW EQGDLERTVLDQQPIPMMDRNKAAELPKLQVGFIDFVCTFVYKE FSRFHEEILPMFDRLQNNRKEWKALADEYEAK hPDE6C MGEINQVAVEKYLEENPQFAKEYFDRKLRVEVLGEIFKNSQVPV 131 (Uniprot ID: QSSMSFSELTQVEESALCLELLWTVQEEGGTPEQGVHRALQRLA P51160) HLLQADRCSMFLCRSRNGIPEVASRLLDVTPTSKFEDNLVGPDKE VVFPLDIGIVGWAAHTKKTHNVPDVKKNSHFSDFMDKQTGYVT KNLLATPIVVGKEVLAVIMAVNKVNASEFSKQDEEVFSKYLNFV SIILRLHHTSYMYNIESRRSQILMWSANKVFEELTDVERQFHKAL YTVRSYLNCERYSIGLLDMTKEKEFYDEWPIKLGEVEPYKGPKTP DGREVNFYKIIDYILHGKEEIKVIPTPPADHWTLISGLPTYVAENGF ICNMMNAPADEYFTFQKGPVDETGWVIKNVLSLPIVNKKEDIVG VATFYNRKDGKPFDEHDEYIIETLTQFLGWSLLNTDTYDKMNKL ENRKDIAQEMLMNQTKATPEEIKSILKFQEKLNVDVIDDCEEKQL VAILKEDLPDPRSAELYEFRFSDFPLTEHGLIKCGIRLFFEINVVEK FKVPVEVLTRWMYTVRKGYRAVTYHNWRHGFNVGQTMFTLLM TGRLKKYYTDLEAFAMLAAAFCHDIDHRGTNNLYQMKSTSPLA RLHGSSILERHHLEYSKTLLQDESLNIFQNLNKRQFETVIHLFEVAI IATDLALYFKKRTMFQKIVDACEQMQTEEEAIKYVTVDPTKKEII MAMMMTACDLSAITKPWEVQSQVALMVANEFWEQGDLERTVL QQQPIPMMDRNKRDELPKLQVGFIDFVCTFVYKEFSRFHKEITPM LSGLQNNRVEWKSLADEYDAKMKVIEEEAKKQEGGAEKAAEDS GGGDDKKSKTCLML hPDE6C DDCEEKQLVAILKEDLPDPRSAELYEFRFSDFPLTEHGLIKCGIRLF 132 Catalytic FEINVVEKFKVPVEVLTRWMYTVRKGYRAVTYHNWRHGFNVG domain (Amino QTMFTLLMTGRLKKYYTDLEAFAMLAAAFCHDIDHRGTNNLYQ acid 483-822 of MKSTSPLARLHGSSILERHHLEYSKTLLQDESLNIFQNLNKRQFET PDE6C) VIHLFEVAIIATDLALYFKKRTMFQKIVDACEQMQTEEEAIKYVT VDPTKKEIIMAMMMTACDLSAITKPWEVQSQVALMVANEFWEQ GDLERTVLQQQPIPMMDRNKRDELPKLQVGFIDFVCTFVYKEFSR FHKEITPMLSGLQNNRVEWKSLADEYDAK hPDE7A MEVCYQLPVLPLDRPVPQHVLSRRGAISFSSSSALFGCPNPRQLSQ 133 (Uniprot ID: RRGAISYDSSDQTALYIRMLGDVRVRSRAGFESERRGSHPYIDFRI Q13946) FHSQSEIEVSVSARNIRRLLSFQRYLRSSRFFRGTAVSNSLNILDDD YNGQAKCMLEKVGNWNFDIFLFDRLTNGNSLVSLTFHLFSLHGLI EYFHLDMMKLRRFLVMIQEDYHSQNPYHNAVHAADVTQAMHC YLKEPKLANSVTPWDILLSLIAAATHDLDHPGVNQPFLIKTNHYL ATLYKNTSVLENHHWRSAVGLLRESGLFSHLPLESRQQMETQIG ALILATDISRQNEYLSLFRSHLDRGDLCLEDTRHRHLVLQMALKC ADICNPCRTWELSKQWSEKVTEEFFHQGDIEKKYHLGVSPLCDR HTESIANIQIGFMTYLVEPLFTEWARFSNTRLSQTMLGHVGLNKA SWKGLQREQSSSEDTDAAFELNSQLLPQENRLS hPDE7A FHLDMMKLRRFLVMIQEDYHSQNPYHNAVHAADVTQAMHCYL 134 Catalytic KEPKLANSVTPWDILLSLIAAATHDLDHPGVNQPFLIKTNHYLAT domain (Amino LYKNTSVLENHHWRSAVGLLRESGLFSHLPLESRQQMETQIGALI acid 187-451 of LATDISRQNEYLSLFRSHLDRGDLCLEDTRHRHLVLQMALKCADI PDE7A) CNPCRTWELSKQWSEKVTEEFFHQGDIEKKYHLGVSPLCDRHTE SIANIQIGFMTYLVEPLFTEWARFSNTRLSQTMLGHVGLNKASW hPDE7B MSCLMVERCGEILFENPDQNAKCVCMLGDIRLRGQTGVRAERRG 135 (Uniprot ID: SYPFIDFRLLNSTTYSGEIGTKKKVKRLLSFQRYFHASRLLRGIIPQ Q9NP56) APLHLLDEDYLGQARHMLSKVGMWDFDIFLFDRLTNGNSLVTLL CHLFNTHGLIHHFKLDMVTLHRFLVMVQEDYHSQNPYHNAVHA ADVTQAMHCYLKEPKLASFLTPLDIMLGLLAAAAHDVDHPGVN QPFLIKTNHHLANLYQNMSVLENHHWRSTIGMLRESRLLAHLPK EMTQDIEQQLGSLILATDINRQNEFLTRLKAHLHNKDLRLEDAQD RHFMLQIALKCADICNPCRIWEMSKQWSERVCEEFYRQGELEQK FELEISPLCNQQKDSIPSIQIGFMSYIVEPLFREWAHFTGNSTLSEN MLGHLAHNKAQWKSLLPRQHRSRGSSGSGPDHDHAGQGTESEE QEGDSP hPDE7B YHNAVHAADVTQAMHCYLKEPKLASFLTPLDIMLGLLAAAAHD 136 Catalytic VDHPGVNQPFLIKTNHHLANLYQNMSVLENHHWRSTIGMLRESR domain (Amino LLAHLPKEMTQDIEQQLGSLILATDINRQNEFLTRLKAHLHNKDL acid 172-410 of RLEDAQDRHFMLQIALKCADICNPCRIWEMSKQWSERVCEEFYR PDE7B) QGELEQKFELEISPLCNQQKDSIPSIQIGFMSYIVEPLFREWAHFTG NSTLSENMLGHLAHNK hPDE8A MGCAPSIHISERLVAEDAPSPAAPPLSSGGPRLPQGQKTAALPRTR 137 (Uniprot ID: GAGLLESELRDGSGKKVAVADVQFGPMRFHQDQLQVLLVFTKE 060658) DNQCNGFCRACEKAGFKCTVTKEAQAVLACFLDKHHDIIIIDHRN PRQLDAEALCRSIRSSKLSENTVIVGVVRRVDREELSVMPFISAGF TRRYVENPNIMACYNELLQLEFGEVRSQLKLRACNSVFTALENSE DAIEITSEDRFIQYANPAFETTMGYQSGELIGKELGEVPINEKKAD LLDTINSCIRIGKEWQGIYYAKKKNGDNIQQNVKIIPVIGQGGKIR HYVSIIRVCNGNNKAEKISECVQSDTHTDNQTGKHKDRRKGSLD VKAVASRATEVSSQRRHSSMARIHSMTIEAPITKVINIINAAQESSP MPVTEALDRVLEILRTTELYSPQFGAKDDDPHANDLVGGLMSDG LRRLSGNEYVLSTKNTQMVSSNIITPISLDDVPPRIARAMENEEYW DFDIFELEAATHNRPLIYLGLKMFARFGICEFLHCSESTLRSWLQII EANYHSSNPYHNSTHSADVLHATAYFLSKERIKETLDPIDEVAALI AATIHDVDHPGRTNSFLCNAGSELAILYNDTAVLESHHAALAFQL TTGDDKCNIFKNMERNDYRTLRQGIIDMVLAIEMTKHFEHVNKF VNSINKPLATLEENGETDKNQEVINTMLRTPENRTLIKRMLIKCA DVSNPCRPLQYCIEWAARISEEYFSQTDEEKQQGLPVVMPVFDRN TCSIPKSQISFIDYFITDMFDAWDAFVDLPDLMQHLDNNFKYWKG LDEMKLRNLRPPPE hPDE8A LHCSESTLRSWLQIIEANYHSSNPYHNSTHSADVLHATAYFLSKE 138 Catalytic RIKETLDPIDEVAALIAATIHDVDHPGRTNSFLCNAGSELAILYND domain (Amino TAVLESHHAALAFQLTTGDDKCNIFKNMERNDYRTLRQGIIDMV acid 531-813 of LATEMTKHFEHVNKFVNSINKPLATLEENGETDKNQEVINTMLR PDE8A) TPENRTLIKRMLIKCADVSNPCRPLQYCIEWAARISEEYFSQTDEE KQQGLPVVMPVFDRNTCSIPKSQISFIDYFITDMFDAWDAFVDLP DLMQHLDNNFKYW hPDE8B MGCAPSIHVSQSGVIYCRDSDESSSPRQTTSVSQGPAAPLPGLFVQ 139 (Uniprot ID: TDAADAIPPSRASGPPSVARVRRARTELGSGSSAGSAAPAATTSR 095263) GRRRHCCSSAEAETQTCYTSVKQVSSAEVRIGPMRLTQDPIQVLLI FAKEDSQSDGFWWACDRAGYRCNIARTPESALECFLDKHHEIIVI DHRQTQNFDAEAVCRSIRATNPSEHTVILAVVSRVSDDHEEASVL PLLHAGFNRRFMENSSIIACYNELIQIEHGEVRSQFKLRACNSVFT ALDHCHEMEITSDDHVIQYVNPAFERMMGYHKGELLGKELADL PKSDKNRADLLDTINTCIKKGKEWQGVYYARRKSGDSIQQHVKI TPVIGQGGKIRHFVSLKKLCCTTDNNKQIHKIHRDSGDNSQTEPHS FRYKNRRKESIDVKSISSRGSDAPSLQNRRYPSMARIHSMTIEAPIT KVINIINAAQENSPVTVAEALDRVLEILRTTELYSPQLGTKDEDPH TSDLVGGLMTDGLRRLSGNEYVFTKNVHQSHSHLAMPITINDVPP CISQLLDNEESWDFNIFELEAITHKRPLVYLGLKVFSRFGVCEFLN CSETTLRAWFQVIEANYHSSNAYHNSTHAADVLHATAFFLGKER VKGSLDQLDEVAALIAATVHDVDHPGRTNSFLCNAGSELAVLYN DTAVLESHHTALAFQLTVKDTKCNIFKNIDRNHYRTLRQAIIDMV LATEMTKHFEHVNKFVNSINKPMAAEIEGSDCECNPAGKNFPEN QILIKRMMIKCADVANPCRPLDLCIEWAGRISEEYFAQTDEEKRQ GLPVVMPVFDRNTCSIPKSQISFIDYFITDMFDAWDAFAHLPALM QHLADNYKHWKTLDDLKCKSLRLPSDS hPDE8B LNCSETTLRAWFQVIEANYHSSNAYHNSTHAADVLHATAFFLGK 140 Catalytic ERVKGSLDQLDEVAALIAATVHDVDHPGRTNSFLCNAGSELAVL domain (Amino YNDTAVLESHHTALAFQLTVKDTKCNIFKNIDRNHYRTLRQABD acid 590-868 of MVLAIEMTKHFEHVNKFVNSINKPMAAEIEGSDCECNPAGKNFP PDE8B) ENQILIKRMMIKCADVANPCRPLDLCIEWAGRISEEYFAQTDEEK RQGLPVVMPVFDRNTCSIPKSQISFIDYFITDMFDAWDAFAHLPAL MQHLADNYKHW hPDE9A MGSGSSSYRPKAIYLDIDGRIQKVIFSKYCNSSDIMDLFCIATGLPR 141 (Uniprot ID: NTTISLLTTDDAMVSIDPTMPANSERTPYKVRPVAIKQLSAGVED 076083) KRTTSRGQSAERPLRDRRVVGLEQPRREGAFESGQVEPRPREPQG CYQEGQRIPPEREELIQSVLAQVAEQFSRAFKINELKAEVANHLA VLEKRVELEGLKVVEIEKCKSDIKKMREELAARSSRTNCPCKYSF LDNHKKLTPRRDVPTYPKYLLSPETIEALRKPTFDVWLWEPNEM LSCLEHMYHDLGLVRDFSINPVTLRRWLFCVHDNYRNNPFHNFR HCFCVAQMMYSMVWLCSLQEKFSQTDILILMTAAICHDLDHPGY NNTYQINARIELAVRYNDISPLENHHCAVAFQILAEPECNIFSNIPP DGFKQIRQGMITLILATDMARHAEIMDSFKEKMENFDYSNEEHM TLLKMILIKCCDISNEVRPMEVAEPWVDCLLEEYFMQSDREKSEG LPVAPFMDRDKVTKATAQIGFIKFVLIPMFETVTKLFPMVEEIML QPLWESRDRYEELKRIDDAMKELQKKTDSLTSGATEKSRERSRD VKNSEGDCA hPDE9A FSINPVTLRRWLFCVHDNYRNNPFHNFRHCFCVAQMMYSMVWL 142 Catalytic CSLQEKFSQTDILILMTAAICHDLDHPGYNNTYQINARIELAVRY domain (Amino NDISPLENHHCAVAFQILAEPECNIFSNIPPDGFKQIRQGMITLILAT acid 288-550 of DMARHAEIMDSFKEKMENFDYSNEEHMTLLKMILIKCCDISNEV PDE9A) RPMEVAEPWVDCLLEEYFMQSDREKSEGLPVAPFMDRDKVTKA TAQIGFIKFVLIPMFETVTKLFPMVEEIMLQPLWESRDRY hPDE10A MRIEERKSQHLTGLTDEKVKAYLSLHPQVLDEFVSESVSAETVEK 143 (Uniprot ID: WLKRKNNKSEDESAPKEVSRYQDTNMQGVVYELNSYIEQRLDT Q9Y233) GGDNQLLLYELSSIIKIATKADGFALYFLGECNNSLCIFTPPGIKEG KPRLIPAGPITQGTTVSAYVAKSRKTLLVEDILGDERFPRGTGLES GTRIQSVLCLPIVTAIGDLIGILELYRHWGKEAFCLSHQEVATANL AWASVAIHQVQVCRGLAKQIELNDFLLDVSKTYFDNIVAIDSLLE HIMIYAKNLVNADRCALFQVDHKNKELYSDLFDIGEEKEGKPVF KKTKEIRFSIEKGIAGQVARTGEVLNIPDAYADPRFNREVDLYTG YTTRNILCMPIVSRGSVIGVVQMVNKISGSAFSKTDENNFKMFAV FCALALHCANMYHRIRHSECIYRVTMEKLSYHSICTSEEWQGLM QFTLPVRLCKEIELFHFDIGPFENMWPGIFVYMVHRSCGTSCFELE KLCRFIMSVKKNYRRVPYHNWKHAVTVAHCMYAILQNNHTLFT DLERKGLLIACLCHDLDHRGFSNSYLQKFDHPLAALYSTSTMEQ HHFSQTVSILQLEGHNIFSTLSSSEYEQVLEIIRKAIIATDLALYFGN RKQLEEMYQTGSLNLNNQSHRDRVIGLMMTACDLCSVTKLWPV TKLTANDIYAEFWAEGDEMKKLGIQPIPMMDRDKKDEVPQGQL GFYNAVAIPCYTTLTQILPPTEPLLKACRDNLSQWEKVIRGEETAT WISSPSVAQKAAASED hPDE10A CKEIELFHFDIGPFENMWPGIFVYMVHRSCGTSCFELEKLCRFIMS 144 Catalytic VKKNYRRVPYHNWKHAVTVAHCMYAILQNNHTLFTDLERKGLL domain (Amino IACLCHDLDHRGFSNSYLQKFDHPLAALYSTSTMEQHHFSQTVSI acid 458-760 of LQLEGHNIFSTLSSSEYEQVLEIIRKAIIATDLALYFGNRKQLEEMY PDE10A) QTGSLNLNNQSHRDRVIGLMMTACDLCSVTKLWPVTKLTANDIY AEFWAEGDEMKKLGIQPIPMMDRDKKDEVPQGQLGFYNAVAIP CYTTLTQILPPTEPLLKACRDNLSQWEKVIRGEE hPDE11A MAASRLDFGEVETFLDRHPELFEDYLMRKGKQEMVEKWLQRHS 145 (Uniprot ID: QGQGALGPRPSLAGTSSLAHSTCRGGSSVGGGTGPNGSAHSQPLP Q9HCR9) GGGDCGGVPLSPSWAGGSRGDGNLQRRASQKELRKSFARSKAIH VNRTYDEQVTSRAQEPLSSVRRRALLRKASSLPPTTAHILSALLES RVNLPRYPPTAIDYKCHLKKHNERQFFLELVKDISNDLDLTSLSY KILIFVCLMVDADRCSLFLVEGAAAGKKTLVSKFFDVHAGTPLLP CSSTENSNEVQVPWGKGIIGYVGEHGETVNIPDAYQDRRFNDEID KLTGYKTKSLLCMPIRSSDGEIIGVAQAINKIPEGAPFTEDDEKVM QMYLPFCGIAISNAQLFAASRKEYERSRALLEVVNDLFEEQTDLE KIVKKIMHRAQTLLKCERCSVLLLEDIESPVVKFTKSFELMSPKCS ADAENSFKESMEKSSYSDWLINNSIAELVASTGLPVNISDAYQDP RFDAEADQISGFHIRSVLCVPIWNSNHQIIGVAQVLNRLDGKPFDD ADQRLFEAFVIFCGLGINNTIMYDQVKKSWAKQSVALDVLSYHA TCSKAEVDKFKAANIPLVSELAIDDIHFDDFSLDVDAMITAALRM FMELGMVQKFKIDYETLCRWLLTVRKNYRMVLYHNWRHAFNV CQLMFAMLTTAGFQDILTEVEILAVIVGCLCHDLDHRGTNNAFQ AKSGSALAQLYGTSATLEHHHFNHAVMILQSEGHNIFANLSSKEY SDLMQLLKQSILATDLTLYFERRIEFFELVSKGEYDWNIKNHRDI FRSMLMTACDLGAVTKPWEISRQVAELVTSEFFEQGDRERLELK LTPSAIFDRNRKDELPRLQLEWIDSICMPLYQALVKVNVKLKPML DSVATNRSKWEELHQKRLLASTASSSPASVMVAKEDRN hPDE11A FKIDYETLCRWLLTVRKNYRMVLYHNWRHAFNVCQLMFAMLT 146 Catalytic TAGFQDILTEVEILAVIVGCLCHDLDHRGTNNAFQAKSGSALAQL Domain YGTSATLEHHHFNHAVMILQSEGHNIFANLSSKEYSDLMQLLKQS (Amino acid ILATDLTLYFERRIEFFELVSKGEYDWNIKNHRDIFRSMLMTACD 640-905 of LGAVTKPWEISRQVAELVTSEFFEQGDRERLELKLTPSAIFDRNRK PDE11A) DELPRLQLEWIDSICMPLYQALVKVNVKLKPMLDSVATNRSKW

In some embodiments, the hPDE5 derived destabilizing domains may be derived from variants, and/or isoforms of hPDE5. The three isoforms hPDE5 Isoform 1, hPDE5 Isoform 2, and hPDE5 Isoform 3 differ at their N terminal regions, and all 3 have unique first exons followed by a common sequence of 823 amino acids. Accordingly, hPDE5 derived DDs may be derived from hPDE5 Isoform 1 (SEQ ID NO. 1; encoded by the nucleotide sequence of SEQ ID NO. 2; hPDE5 Isoform 2 (SEQ ID NO. 147; encoded by the nucleotides 113-2611 of the nucleotide sequence of SEQ ID NO. 148 or the nucleotide sequence of SEQ ID NO. 380) and/or hPDE5 Isoform 3 (SEQ ID NO. 149; encoded by the nucleotides 95-2563 of nucleotide sequence of SEQ ID NO. 150 or SEQ ID NO. 381). In some embodiments, the hPDE5 DDs may be derived from hPDE5 Isoform X1 (SEQ ID NO. 382).

In some embodiments, the DD mutations identified herein may be mapped back to the hPDE5 sequence to identify DD hotspots. DD hotspots as used herein refer to amino acids within the hPDE5 of SEQ ID NO. 1 whose mutation results in the “responsive” nature of the stimulus responsive element generated from hPDE5. The DD characteristics may be improved by saturation mutagenesis, which involves mutating the amino acids at the hotspot position to any of the known amino acids, including, but not limited to lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan, alanine, isoleucine, leucine, phenylalanine, valine, proline, and glycine. In some instances, a library of hotspot mutations may be generated by site directed mutagenesis and each of the mutants in the library is fused to a reporter protein e.g. AcGFP (SEQ ID NO. 79) via a linker such as SG. The properties of the DDs may be analyzed in the presence and absence of ligands such as Sildenafil, Vardenafil and Tadalafil. In some embodiments, the arginine at position 732 (also referred to as R732) of the hPDE5 of SEQ ID NO. 1, may be mutated to any of the known amino acids and are provided in Table 5A. The mutations of the amino acid R732 may include but are not limited to R732L, R732A, R732G, R732V, R732I, R732P, R732F, R732W, R732Y, R732H, R732S, R732T, R732D, R732E, R732Q, R732N, R732M, R732C, and R732K. The hPDE5 mutants fused either at the N terminus or the C terminus to a linker and GFP are provided in Table 6. In Table 5A, the mutations are in bold. Table 5B provides the additional components that may be combined with the mutants listed in Table 5A to generate the constructs described in Table 6. In Table 6, asterisk indicates the translation of the stop codon. Table 6 also provides alternate aliases for a given construct ID. These aliases are identified by the prefix OT.

TABLE 5A hPDE5 R732 mutants Construct AA NA ID/ SEQ ID SEQ ID Description Sequence NO. NO. hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 151 169 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRGGEFFELIRKNQFNLEDPHQKEL R732G) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 152 170 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRAGEFFELIRKNQFNLEDPHQKEL R732A) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 153 171 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRVGEFFELIRKNQFNLEDPHQKEL R732V) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 154 172 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, R732I) TLKIIKQAILATDLALYIKRIGEFFELIRKNQFNLEDPHQKELF LAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKE LNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 155 173 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, R732P) TLKIIKQAILATDLALYIKRPGEFFELIRKNQFNLEDPHQKEL FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 156 174 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, R732F) TLKIIKQAILATDLALYIKRFGEFFELIRKNQFNLEDPHQKEL FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 157 175 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRWGEFFELIRKNQFNLEDPHQKEL R732W) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 158 176 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRYGEFFELIRKNQFNLEDPHQKEL R732Y) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 159 177 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRHGEFFELIRKNQFNLEDPHQKEL R732H) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 160 178 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, R732S) TLKIIKQAILATDLALYIKRSGEFFELIRKNQFNLEDPHQKEL FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 161 179 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, R732T) TLKIIKQAILATDLALYIKRTGEFFELIRKNQFNLEDPHQKEL FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 162 180 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRDGEFFELIRKNQFNLEDPHQKEL R732D) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 163 181 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, R732E) TLKIIKQAILATDLALYIKREGEFFELIRKNQFNLEDPHQKEL FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 164 182 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRQGEFFELIRKNQFNLEDPHQKEL R732Q) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 165 183 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRNGEFFELIRKNQFNLEDPHQKEL R732N) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 166 184 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRMGEFFELIRKNQFNLEDPHQKEL R732M) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 167 185 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, R732C) TLKIIKQAILATDLALYIKRCGEFFELIRKNQFNLEDPHQKEL FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA 168 186 (Amino acid ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSE 590-836 of HPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKT WT, TLKIIKQAILATDLALYIKRKGEFFELIRKNQFNLEDPHQKEL R732K) FLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERK ELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 383 401; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 402 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732G) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRGGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 384 403; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 404 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732A) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRAGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR  12 405 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, R732L) RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN QILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 385 406; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 407 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732V) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRVGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 386 408 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, R732I) RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN QILSGLSIEEYKTTLKIIKQAILATDLALYIKRIGEFFELIRKNQ FNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVTE FFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQ LYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 387 409; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 410 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, R732P) RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN QILSGLSIEEYKTTLKIIKQAILATDLALYIKRPGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 388 411 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, R732F) RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN QILSGLSIEEYKTTLKIIKQAILATDLALYIKRFGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 389 412 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732W) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRWGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 390 413; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 414 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732Y) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRYGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 391 415; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 416 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732H) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRHGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 392 417; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 418 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, R732S) RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN QILSGLSIEEYKTTLKIIKQAILATDLALYIKRSGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 393 419 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of  HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, R732T) RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN QILSGLSIEEYKTTLKIIKQAILATDLALYIKRTGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 394 420; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 421 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732D) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRDGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 395 422; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 423 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, R732E) RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN QILSGLSIEEYKTTLKIIKQAILATDLALYIKREGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 396 424; (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 425 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732Q) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRQGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 397 426 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732N) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRNGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 398 427 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732M) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRMGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 399 428 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, R732C) RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN QILSGLSIEEYKTTLKIIKQAILATDLALYIKRCGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 400 429 (Amino acid MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWR 535-860 of HAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDH WT, RGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGN R732K) QILSGLSIEEYKTTLKIIKQAILATDLALYIKRKGEFFELIRKN QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVA TEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ

TABLE 5B Additional hPDE5 R732 construct components AA SEQ NA SEQ Component Amino Acid Sequence ID NO ID NO AcGFP VSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDA  79 372 (Amino acid TYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRY 2-239 of WT) PDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRA EVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYN AHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMI YFGFVTAAAITHGMDELYK AcGFP MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGD 365 373 (Amino acid ATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSR 1-239 of WT) YPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYKSR AEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNY NAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADH YQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH MIYFGFVTAAAITHGMDELYK linker (SG) SG — AGTGGT linker (GS) GS — GGATCC

TABLE 6 hPDE5 R732 constructs AA NA SEQ SEQ Construct ID/ ID ID Description Sequence NO. NO. OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 187 205 037 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001233, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-037) QAILATDLALYIKRGGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732G); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 188 206 038 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001234, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-038) QAILATDLALYIKRAGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732A); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 189 207 039 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001235, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-039) QAILATDLALYIKRVGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732V); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 190 208 040 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001236, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-040) QAILATDLALYIKRIGEFFELIRKNQFNLEDPHQKELFLAMLMT hPDE5 ACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLM (Amino acid NREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFTGI 590-836 of VPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLP WT, R732I); VPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQER linker (SG); TIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGN AcGFP KMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLAD (Amino acid 2- HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGF 239 of WT); VTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 191 209 041 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001237, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-041) QAILATDLALYIKRPGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732P); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 192 210 042 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001238, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-042) QAILATDLALYIKRFGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732F); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 193 211 043 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001239, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-043) QAILATDLALYIKRWGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732W); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 194 212 044 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001240, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-044) QAILATDLALYIKRYGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732Y); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 195 213 045 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001241, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-045) QAILATDLALYIKRHGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732H); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 196 214 046 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001242, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-046) QAILATDLALYIKRSGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732S); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 197 215 047 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001243, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-047) QAILATDLALYIKRTGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732T); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 198 216 048 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001244, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-048) QAILATDLALYIKRDGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732D); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 199 217 049 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001245, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-049) QAILATDLALYIKREGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732E); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 200 218 050 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001246, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-050) QAILATDLALYIKRQGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732Q); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 201 219 051 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001247, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-051) QAILATDLALYIKRNGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732N); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 202 220 052 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001248, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-052) QAILATDLALYIKRMGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732M); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 203 221 053 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001249, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-053) QAILATDLALYIKRCGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732C); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5N- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAAL 204 222 054 (OT- KAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPL 001250, OT- AQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIK hPDE5-054) QAILATDLALYIKRKGEFFELIRKNQFNLEDPHQKELFLAMLM hPDE5 TACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL (Amino acid MNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFT 590-836 of GIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL WT, R732K); PVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQE linker (SG); RTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG AcGFP NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA (Amino acid 2- DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF 239 of WT); GFVTAAAITHGMDELYK* stop OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 430 467 064 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001186) AFNTAQCMFAALKAGKIQNKLTDLEILALLTAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRGGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732G); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 431 468 065 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001187) AFNTAQCMFAALKAGKIQNKLTDLEILALLTAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRAGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732A); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 432 469 066 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001188) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRVGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732V); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 433 470 067 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001189) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRIGEFFELIRKNQFNLED (Amino acid PHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGD 535-860 of RERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTH WT, R732I); VSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVPIL linker (SG); IELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP AcGFP TLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFE (Amino acid 2- DDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEY 239 of WT); NYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQ stop NTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAA AITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 434 471 068 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001190) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRPGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732P); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 435 472 069 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001191) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRFGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732F); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 436 473 070 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001192) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRWGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732W); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 437 474 071 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001193) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRYGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732Y); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 438 475 072 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001195) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRHGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732H); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 439 476 073 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001196) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRSGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732S); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 440 477 074 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001197) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRTGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732T); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 441 478 075 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001198) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRDGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732D); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 442 479 076 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001199) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKREGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732E); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 443 480 077 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001200) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRQGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732Q); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 444 481 078 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001201) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRNGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732N); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 445 482 079 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001202) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRMGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732M); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 446 483 080 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001203) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRCGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732C); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIR 447 484 081 (OT- MFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRH 001204) AFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRG Methionine; VNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS hPDE5 GLSIEEYKTTLKIIKQAILATDLALYIKRKGEFFELIRKNQFNLE (Amino acid DPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQG 535-860 of DRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT WT, R732K); HVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIVP linker (SG); ILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP AcGFP WPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF (Amino acid 2- FEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKM 239 of WT); EYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ stop QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTA AAITHGMDELYK* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 448 485 096 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001279) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732G); ATDLALYIKRGGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 449 486 097 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001280) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732A); ATDLALYIKRAGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 450 487 098 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001281) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732L); ATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 451 488 099 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001282) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732M); ATDLALYIKRMGEFFELIRKNQFNLEDPHQKELFLAMLMTAC Xba I site DLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNR (TCTAGA); EKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR stop QKWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 452 489 100 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001283) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732F); ATDLALYIKRFGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 453 490 101 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001284) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732W); ATDLALYIKRWGEFFELIRKNQFNLEDPHQKELFLAMLMTAC Xba I site DLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNR (TCTAGA); EKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR stop QKWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 454 491 102 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001285) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732K); ATDLALYIKRKGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 455 492 103 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001286) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732Q); ATDLALYIKRQGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 456 493 104 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001287) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732E); ATDLALYIKREGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 457 494 105 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001288) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732S); ATDLALYIKRSGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 458 495 106 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001289) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732P); ATDLALYIKRPGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 459 496 107 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001290) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732V); ATDLALYIKRVGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 460 497 108 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001291) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732I); ATDLALYIKRIGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 461 498 109 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001292) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732C); ATDLALYIKRCGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 462 499 110 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001293) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732Y); ATDLALYIKRYGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 463 500 111 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001294) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732H); ATDLALYIKRHGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 464 501 112 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001295) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732N); ATDLALYIKRNGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 465 502 113 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001296) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732D); ATDLALYIKRDGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKL 466 503 114 (OT- TLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFK 001297) SAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGT AcGFP DFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRH (Amino acid 1- NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN 239 of WT); EKRDHMIYFGFVTAAAITHGMDELYKGSEETRELQSLAAAVV linker (GS); PSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH hPDE5 EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAG (Amino acid KIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLY 535-860 of CHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732T); ATDLALYIKRTGEFFELIRKNQFNLEDPHQKELFLAMLMTACD Xba I site LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNRE (TCTAGA); KKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ stop KWQALAEQQSR*

In some embodiments, any of the mutations taught in Tables 1, 2, and 5A may be combined. In one embodiment, the combination mutations are provided in Table 7. Combination mutations may be linked to AcGFP (Amino acid 2-239 of WT) (SEQ ID NO. 79); encoded by the nucleotide sequence of SEQ ID NO. 372; AcGFP (Amino acid 1-239 of WT) (SEQ ID NO. 365); encoded by the nucleotide sequence of SEQ ID NO. 94; or firefly luciferase, Fluc (N50D, N119G, S548I, K549A, L550V) (SEQ ID NO. 223) encoded by the nucleotide sequence of SEQ ID NO. 224. In some embodiments, the portions of the construct may be linked through a linker e.g. SG linker, encoded by the nucleotide sequence (AGTGGT). In some embodiments, the constructs described in Table 7 may comprise an Xba I restriction site, SR, encoded by the nucleotide sequence, TCTAGA. Table 7 provides the constructs comprising the combination mutations. In Table 7, translation of the stop codon is indicated by asterisk. Also provided in Table7 are alternate aliases for a given construct ID. These aliases are identified by the prefix OT.

TABLE 7 hPDE5 combination mutations AA NA Construct SEQ SEQ ID/ ID ID Description Sequence NO. NO. hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCT 504 519 (Amino acid IRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH 535-860 of NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS WT, F736A, HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLM D764N) ILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRG EFAELIRKNQFNLEDPHQKELFLAMLMTACNLSAITKPW PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNK IPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQK WQALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL 225 231 hPDE5 CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQC WT, F736A, LMILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKR D764N) RGEFAELIRKNQFNLEDPHQKELFLAMLMTACNLSAITKP WPIQQRIAELVA1EFFDQGDRERKELNIEPTDLMNREKKN KIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ KWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCT 505 520 (Amino acid IRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH 535-860 of NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS WT, R732L, HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLM D764N) ILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG EFFELIRKNQFNLEDPHQKELFLAMLMTACNLSAITKPWP IQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQK WQALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL 226 232 hPDE5 CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQC WT, R732L, LMILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKR D764N) LGEFFELIRKNQFNLEDPHQKELFLAMLMTACNLSAITKP WPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKN KIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ KWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 227 233 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, R732L, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL F736A) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEF AELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQ QRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 506 521 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAFHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, Y612F, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL R732L) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 507 522 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAWH 535-860 of NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS WT, Y612W, HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMI R732L) LNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEF FELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQ QRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 508 523 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAAHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, Y612A, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL R732L) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 509 524 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSA WT, H653A, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL R732L) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFF ELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 510 525 (Amino acid RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHN 535-860 of WRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSH WT, R732L, DLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMIL D764A) NSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFF ELIRKNQFNLEDPHQKELFLAMLMTACALSAITKPWPIQQ RIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSM QVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQA LAEQQ OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 228 234 025 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001222, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-025) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR hPDE5 GEFAELIRKNQFNLEDPHQKELFLAMLMTACNLSAITKPW (Amino acid PIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKI 535-860 of PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW WT, F736A, QALAEQQSGVSKGAELFTGIVPILIELNGDVNGHKFSVSGE D764N); GEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFS linker (SG); RYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAE AcGFP VKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNV (Amino acid YIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD 2-239 of GPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAIT WT); stop HGMDELYK* OT-hPDE5N- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 229 235 026 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001223, OT- HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL hPDE5-026) SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL Methionine; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG hPDE5 EFFELIRKNQFNLEDPHQKELFLAMLMTACNLSAITKPWPI (Amino acid QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS 535-860 of MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ WT, R732L, ALAEQQSGVSKGAELFTGIVPILIELNGDVNGHKFSVSGEG D764N); EGDATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSR linker (SG); YPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEV AcGFP KFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVY (Amino acid IMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDG 2-239 of PVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITH WT); stop GMDELYK* OT- MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGT 230 236 hPDE5C-035 IAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIV (OT-001231, VCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMG OT-hPDE5- ISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGF 035) QSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGST Fluc (N50D, GLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFH N119G, HGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQS S548I, ALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGE K549A, AVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKV L550V); VPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNN linker (SG); PEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKY hPDE5 KGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAV (Amino acid VVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVP 535-860 of KGLTGKLDARKIREILIKAKKGGKIAVSGEETRELQSLAA WT, R732L; AVVPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQ F736A); stop NFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQ CMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNS YIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGL SIEEYKTTLKIIKQAILATDLALYIKRLGEFAELIRKNQFNL EDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEF FDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICL QLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ* OT-hPDE5- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALC 511 526 029 (OT- TIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAY 001225) HNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL Methionine; SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL hPDE5 MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG (Amino acid EFAELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWP 535-860 of IQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIP WT, R732L, SMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW F736A); QALAEQQSGVSKGAELFTGIVPILIELNGDVNGHKFSVSGE Linker (SG); GEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFS AcGFP RYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAE (Amino acid VKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNV 2-239 of YIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD WT); stop GPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAIT HGMDELYK* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 512 527 030 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001226) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); PDE5 LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, R732L, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG F736A); stop EFAELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWP IQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIP SMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKW QALAEQQ* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 513 528 083 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001205) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); hPDE5 LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid FHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, Y612F, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG R732L); stop EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 514 529 084 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001206) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); hPDE5 LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid WHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, Y612W, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG R732L); stop EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 515 530 085 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001207) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); hPDE5 LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid AHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, Y612A, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG R732L); stop EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 516 531 090 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001212) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); hPDE5 LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSADLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, H653A, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG R732L); stop EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 517 532 091 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001213) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); hPDE5 LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA (Amino acid YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA 535-860 of LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, R732L, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG D764A); stop EFFELIRKNQFNLEDPHQKELFLAMLMTACALSAITKPWPI QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ* OT-hPDE5- MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATY 518 533 094 (OT- GKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK 001253) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTL AcGFP VNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK (Amino acid NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN 1-239 of HYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY WT); linker KSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETA (SG); LCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVA hPDE5 (Amino YHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA acid 535- LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL 860 of WT; MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLG R732L); Xba EFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPI I site QQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS (TCTAGA); MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ stop ALAEQQ SR*

In some embodiments, any of the mutations described herein may be tested in the context of the truncated hPDE5 domains. As used herein truncated hPDE5 domains refers to regions and/or portions of hPDE5 of SEQ ID NO. 1, and are exemplified in Table 8. Also provided in Table 8 are the constructs utilizing the regions or portions of hPDE5, fused to AcGFP via linkers. In Table 8, translation of the stop codon is indicated by asterisk. Table 8 also provides alternate aliases for a given construct ID. These aliases are identified by the prefix OT.

TABLE 8 hPDE5 truncation constructs and its components AA NA Construct SEQ SEQ ID/ ID ID Description Sequence NO NO Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRM 237   4 hPDE5 FTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFN (Amino acid TAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY 535-860 of IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK WT) TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRM 238 250 hPDE5 FTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFN (Amino acid TAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY 535-860 of IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK WT, R732L) TTLKIIKQAILATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ Methionine; MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRM 239 251 hPDE5 FTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFN (Amino acid TAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY 535-836 of IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK WT, R732L) TTLKIIKQAILATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS Methionine; MSDLETALCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRK 240 252 hPDE5 NVAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL (Amino acid SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNS 567-860 of PGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRKN WT, R732L) QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFF DQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEA LTHVSEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALK 241 253 (Amino acid AGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQL 590-860 of YCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732L) ATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAMLMTACDL SAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ hPDE5 MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALK 242 254 (Amino acid AGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQL 590-836 of YCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL WT, R732L) ATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAMLMTACDL SAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK NKIPSMQVGFIDAICLQLYEALTHVS hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT   3 339 (Amino acid DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT 535-860 of AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI WT) QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT  12 536 (Amino acid DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT 535-860 of AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI WT, R732L) QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG CRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFT 534 537 (Amino acid DLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNT 535-836 of AQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYI WT, R732L) QRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK TTLKIIKQAILATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFL AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVS hPDE5 SDLETALCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKN 535 538 (Amino acid VAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS 567-860 of HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSP WT, R732L) GNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRKNQ FNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVTEFFD QGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALT HVSEDCFPLLDGCRKNRQKWQALAEQQ Linker (SG) SG — AGT GGT AcGFP VSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLK  79 372 FICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMP EGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDG NILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQ LADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF GFVTAAAITHGMDELYK OT- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRM 243 255 hPDE5N- FTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFN 019 (OT- TAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY 001216, OT- IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK hPDE5-019) TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFL Methionine; AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP hPDE5 TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG (Amino acid CRKNRQKWQALAEQQSGVSKGAELFTGIVPILIELNGDVNGHKF 535-860 of SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCF WT); linker SRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFE (SG); GDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK AcGFP NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS (Amino acid TQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELYK* 2-239 of WT); stop OT- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRM 244 256 hPDE5N- FTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFN 020 (OT- TAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY 001217, OT- IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK hPDE5-020) TTLKIIKQAILATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFL Methionine; AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP hPDE5 TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDG (Amino acid CRKNRQKWQALAEQQSGVSKGAELFTGIVPILIELNGDVNGHKF 535-860 of SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCF WT, SRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFE R732L); GDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAK linker (SG); NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS AcGFP; TQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELYK* stop (Amino acid 2-239 of WT); stop OT- MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRM 245 257 hPDE5N- FTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFN 021 (OT- TAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY 001218, OT- IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYK hPDE5-021) TTLKIIKQAILATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFL Methionine; AMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP hPDE5 TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAEL (Amino acid FTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK 535-836 of LPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQER WT, TIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNK R732L); MEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQ linker (SG); QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAA AcGFP AITHGMDELYK* (Amino acid 2-239 of WT); stop OT- MSDLETALCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRK 246 258 hPDE5N- NVAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAAL 022 (OT- SHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNS 001219, OT- PGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRKN hPDE5-022) QFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFF Methionine; DQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEA hPDE5 LTHVSEDCFPLLDGCRKNRQKWQALAEQQSGVSKGAELFTGIV (Amino acid PILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW 567-860 of PTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFED WT, DGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNY R732L); NAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIG linker (SG); DGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGM AcGFP DELYK* (Amino acid 2-239 of WT); stop OT- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALK 247 259 hPDE5N- AGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQL 023 (OT- YCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL 001220, OT- ATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAMLMTACDL hPDE5-023) SAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK hPDE5 NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQ (Amino acid ALAEQQSGVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGD 590-860 of ATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQ WT, HDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIEL R732L); TGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIR linker (SG); HNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN AcGFP EKRDHMIYFGFVTAAAITHGMDELYK* (Amino acid 2-239 of WT); stop OT- MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALK 248 260 hPDE5N- AGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQL 024 (OT- YCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL 001221, OT- ATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAMLMTACDL hPDE5-024) SAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK hPDE5 NKIPSMQVGFIDAICLQLYEALTHVSSGVSKGAELFTGIVPILIEL (Amino acid NGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVT 590-836 of TLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNY WT, KSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHN R732L); VYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPV linker (SG); LLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY AcGFP K* (Amino acid 2-239 of WT); stop

Stimulus

Biocircuits of the invention are triggered by one or more stimuli. Stimuli may be selected from a ligand, an externally added or endogenous metabolite, the presence or absence of a defined ligand, pH, temperature, light, ionic strength, radioactivity, cellular location, subject site, microenvironment, the presence or the concentration of one or more metal ions.

In some embodiments, the stimulus is a ligand. Ligands may be nucleic acid-based, protein-based, lipid based, organic, inorganic or any combination of the foregoing. In some embodiments, the ligand is selected from the group consisting of a protein, peptide, nucleic acid, lipid, lipid derivative, sterol, steroid, metabolite derivative and a small molecule. In some embodiments, the stimulus is a small molecule. In some embodiments, the small molecules are cell permeable. Ligands useful in the present invention include without limitation, any of those taught in Table 2 of co-owned U.S. Provisional Patent Application No. 62/320,864 filed Apr. 11, 2016, 62/466,596 filed Mar. 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety). In some embodiments, the small molecules are FDA-approved, safe and orally administered.

In some embodiments, the ligand binds to phosphodiesterases. In some embodiments, the ligand binds to and inhibits phosphodiesterase function and is herein referred to as a phosphodiesterase inhibitor.

In some embodiments, the ligand is a small molecule that binds to phosphodiesterase 5. In one embodiment, the small molecule is a hPDE5 inhibitor. Examples of hPDE5 inhibitors include, but are not limited to, Sildenafil, Vardenafil, Tadalafil, Avanafil, Lodenafil, Mirodenafil, Udenafil, Benzamidenafil, Dasantafil, Beminafil, SLx-2101, LAS 34179, UK-343,664, UK-357903, UK-371800, and BMS-341400.

In some embodiments, ligands include sildenafil-derived ligands containing portions of the ligand known to mediate binding to hPDE5. Ligands may also be modified to reduce off-target binding to Phosphodiesterases and increase specific binding to hPDE5.

hPDE5 inhibitors cover a broad pharmacokinetic space with respect to the approved dose and their duration of action and is described in Table 9. In Table 9, PO stands for per os (i.e. by mouth); QD represents quaque die (i.e. every day); IV represents intravenous; TID represents ter un die (i.e. three times a day); and Cmax represents the peak serum concentration that a drug achieves after its administration.

TABLE 9 Pharmacokinetics of hPDE5 inhibitors Duration Drug Approved Dose Cmax of action Sildenafil PO: 100 mg QD or PO: 1 μM 4-8 hrs 20 mg TID IV: 1 μM IV: 10 mg TID Vardenafil PO: 20 mg QD 0.1 μM 2-8 hrs Tadalafil PO: 40 QD 1.5 μM 24-36 hrs 

In some embodiments, the ligand selection is determined by the magnitude and duration of expression of the effector modules of the invention using the PK parameters described in Table 9. In some embodiments, high levels of expression of the payload for a short duration of time may be desired. In such instances, vardenafil may be selected as the ligand. In some embodiments, high levels of expression of the payload may be desired for a long duration. In such instances, the ligand, Tadalafil may be selected. In some embodiments, low levels of expression of the payload may be desired for a long duration of time. In such instances, Sildenafil may be selected as the ligand.

In some embodiments, additional hPDE5 inhibitors may be developed to show selectivity towards a specific phosphodiesterase protein; to reduce or increase the duration of treatment with the ligand; and to improve the rate of onset of the ligand.

Ligands may also be selected from the analysis of the dependence of a known hPDE5 ligand's activity on its molecular/chemical structure, through Structure Activity Relationships (SAR) study. Any of the methods related to SAR, known in art may be utilized to identify stabilizing ligands of the invention. SAR may be utilized to improve properties of the ligand such as specificity, potency, pharmacokinetics, bioavailability, and safety. SAR analysis of known hPDE5 inhibitors may also be combined with high resolution X ray structures of hPDE5 complexed with ligands. The X ray structure of hPDE5 co-crystallized with Sildenafil, Tadalafil, and Vardenafil have been studied and the binding mode of the inhibitors has been identified (Zhang, K. Y. J. et al. (2004) Mol. Cell, 15, 279; Card, G. L. et al. (2004) Structure. 12, 2233; the contents of each of which are incorporated herein by reference in their entirety). There are several classes of hPDE5 inhibitors described. These include aryl, beryl, heteroaryl or heterobiaryl classes with different scaffold structures. The aryl class includes substituted nitroanilines and the biaryl class includes substituted naphthalenes. The heterobiaryl and heterotriaryl are further sub classified based on its fused system into pyrazolopyrimidinones, triazolopyrimidinones, imidazotriazines, purines, pyrrolopyrimidinones, triazolotrizinones, isoxazolopyrimidinones, β-carbolines, pyrroloquinolones, isoquinolines, quinazolines, imidazoquinazolinones, pyrazolopyridines, pyrazolopyridopyrimidinones. These widely different chemical structures are suggested to have different orientation in the binding site of hPDE5 enzyme. Sildenafil has three main chemical groups, the pyrazolopyrimidinone ring, the ethoxyphenyl ring and the methylpiperazine ring. The pyrazolopyrimidinone group is responsible for the binding of the drug to its active binding site of hPDE5.

Many hPDE inhibitors act by competing with the substrate, cGMP, for the catalytic site of the enzyme. Sildenafil and Vardenafil differ in the heterocyclic ring system used to mimic the purine ring of cGMP. They also differ in the substituent (ethyl/methyl) of a piperazine side chain. Although these are the only structural differences, Vardenafil is more potent than Sildenafil. In some embodiments, the structural differences between different know inhibitors of hPDE may be utilized to design better hPDE inhibitor based ligands. For example, Corbin et al. synthesized an analog of sildenafil that contained the sildenafil ring system but with the appended ethyl group found in vardenafil; and an analog of vardenafil (dimethyl-vardenafil) that contained the vardenafil ring system but with the appended methyl group found in sildenafil was also generated. These studies identified that the ring systems play a critical role in higher potency of vardenafil over sildenafil (Corbin J D et al. (2004) Neurochem Int; 45(6):859-63; the contents of which are incorporated herein by reference in their entirety). Based on the X ray crystal structure of hPDE complexed with sildenafil, the active site region was defined as a sphere within the 6.5 Angstrom from the reference ligand, sildenafil. Surface hydrophobicity (lipophilicity) potential physicochemical property map of the hPDE5 active site may be generated based on this information. The ethoxyphenyl group of sildenafil fits into the hydrophobic pocket formed by Phe 786, Ala783, Leu804, and Va1782, and the pyrazolopyrimidinone ring also forms hydrophobic interaction with the side chains of Va1782, Tyr612, and Phe820 in the binding pocket.

New hPDE5 inhibitors may also be developed using a series of analogs of known hPDE5 inhibitors. 3D-Quantitative Structure activity analysis (QSAR), CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) may be implemented for this purpose. In one embodiment, the structure of hPDE5 inhibitor may preferentially incorporate an N ethyl group in the pyrrolopyrimidinone ring at N5 position; and a propoxyphenyl group, which bear a substituent; one methylene unit longer, respectively in comparison with the corresponding positions of sildenafil. Any of the modifications to Sildenafil structure taught by Koo J et al. may be useful in the present invention (Koo J et al. (2007) Bioorg. & Med. Chem Lett 17; 4271-4274; the contents of which are incorporated by reference in their entirety).

In some embodiments, the stimulus may be a ligand that binds to more than one phosphodiesterase. In one embodiment, the stimulus is a pan phosphodiesterase inhibitor that may bind to two or more hPDEs such as Aminophyline, Paraxanthine, Pentoxifylline, Theobromine, Dipyridamole, Theophyline, Zaprinast, Icariin, CDP-840, Etazolate and Glaucine.

In some embodiments, the ligand is a hPDE1 inhibitor. Exemplary hPDE1 inhibitor, Vinpocetine may in some instances be used as the ligand in the present invention. In some embodiments, the ligand is a hPDE2 inhibitor. Exemplary hPDE2 inhibitors include EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), Oxindole, PDP and BAY 60-7550. Inhibitors that selectively inhibit a hPDE2 isoform may also be used, for example, substituted pyrido (2,3-b) pyrazines having a hPDE2A selective inhibitory action (See, e.g., U.S. Pat. No. 9,527,841; the contents of which are incorporated by reference in its entirety). In some embodiments, the ligand is a hPDE3 inhibitor. hPDE3 inhibitors useful in the invention include, but are not limited to Amrinone, Cilostazol, Milrinone, Enoximone, and Pimobendan. In some embodiments, the ligand is a hPDE4 inhibitor. Exemplary hPDE4 inhibitors include FDA approved small molecules such as, but not limited to AN2728 (4-[(1-hydroxy-1,3-dihydro-2,1-benzoxaborol-5-yl) oxy]benzonitrile), Apremilast/CC10004 (N-{2-[(1S)-1-(3-Ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}acetamide), and Roflumilast. Other exemplary hPDE4 inhibitors include Other small molecules that inhibit hPDE4 also include E6005/RVT501, Cilomilast/SB-207,499, Ibudilast (AV-411 or MN-166), Mesembrenone, Piclamilast/RP 73401, Rolipram, Atizoram/CP-80633, Arofylline, CC-1088, Catramilast, CGH-2466, Cipamfylline, Drotaverine, Filaminast/WAY-PDA 641, HT-0712, DNS-001, ICI-63197, Indimilast, Irsogladine/MN 1695, Lirimilast/BAY 19-8004, Oglemilast, Revamilast, Ro 20-1724, Ronomilast, GSK256066, DC-TA-46, AWD 12-281 and YM-976. Any of the hPDE4 inhibitors described in International Patent Publication No. WO2014078220A1 and US Patent Publication No. US20170129887A1 may be useful in the present invention (the contents of each of which are incorporated by reference in their entirety). In some embodiments, the ligand is a hPDE6 inhibitor. In some embodiments, the ligand is a hPDE7 inhibitor. Exemplary hPDE7 inhibitors, include BRL-50481(N, N,2-Trimethyl-5-nitrobenzenesulfonamide) and ASB16165 (1H-Thieno(2,3-C) pyrazole-5-carboxamide, 1-cyclohexyl-N-(6-(4-hydroxy-1-piperidinyl)-3-pyridinyl)-3-methyl). In some embodiments, the ligand is a hPDE8 inhibitor such as PF-04957325 (Pfizer). In some embodiments, the ligand is a hPDE9 inhibitor. Exemplary hPDE9 inhibitors include, but are not limited to BAY73-6691 (1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d] pyrimidine-4-one), PF-04447943 (6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(oxan-4-yl)-2H-pyrazolo[3,4-d]pyrimidin-4-one) and WYQ-C28L. In some embodiments, the ligand is a hPDE10 inhibitor. Exemplary PDE10 inhibitors include, but are not limited to OMS 824, Papaverine and PF-2545920 (2-(4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl) phenoxymethyl) quinolone), and GS-5759 (Gilead).

In one embodiment, the stimuli of the present invention may be FDA approved ligands capable of binding to the specific DDs or target regions within the DDs. In other embodiments, FDA approved ligands may be used to screen potential binders in the human protein. DDs may be designed based on the positive hits from the screen using the portion of the protein that binds to the ligand. In one embodiment, proteins that bind to FDA approved ligands as off target interactions may be used to design DDs of the present invention.

In some embodiments, ligands that do not affect the activity of the immune cell, and/or the chimeric antigen receptor, in the absence of the SREs may be preferably selected.

Stabilizing Domains

In some embodiments, the stimulus response element may be stabilized in the absence of the stimulus but destabilized by the stimulus. In some embodiments, SREs may be derived from protein complexes that comprise at least one protein-protein interaction. In other aspects, the SRE may form a protein-protein interaction with a natural protein within the cell. Protein complexes reduce the exposure of the constituent proteins to the risk of undesired oligomerization by reducing the concentration of the free monomeric state. Payloads appended to such SREs may stabilized in the absence of the stimulus. In some aspects, the stimulus may be a small molecule that is capable of interrupting or disrupting the protein-protein interactions related to the SRE. In such instances, addition of the stimulus, results in the reduced expression and/or function of the payload. In some embodiments, stimuli that induce conformational change of the SRE may be utilized. In one aspect, the SRE may be stabilized by the conformational change. In another aspect, the SRE may be destabilized by the conformational change. The stimuli may also be small molecules that disrupt post translational modification of SREs which may result in the disruption of the protein-protein interaction related to the SRE. In some embodiments, SREs may be identified using protein interactomic techniques known in the art. Such methods may enable the identification of protein interactions that are therapeutically relevant. Any of the large-scale quantitative proteomics methods described in International Patent Publication NOs. WO2017210427A1, WO2016196994A9, and WO2014200987A3 may be useful in the present invention (the contents of each of which are incorporated by reference in their entirety).

Payloads

In some embodiments, payloads can be any natural protein in an organism genome, a fusion polypeptide, an antibody, or variants, mutants and derivatives thereof. In some embodiments, the effector module of the present invention is a fusion construct comprising a DD of the invention operably linked to at least one payload. In one aspect, the payload may be any natural protein of interest (POI) or variants thereof, an antibody or fragments thereof, a therapeutic agent, or any artificial peptide or polypeptide.

In some embodiments, payloads of the present invention may be immunotherapeutic agents. As used herein, an immunotherapeutic agent is any agent that induces immune responses in an organism. An immunotherapeutic agent may be a natural protein in an organism or it may be an artificial protein such as a fusion protein or an antibody. The immunotherapeutic agent may be, but is not limited to, an antibody and fragments and variants thereof, a MEW molecule, an antigen and fragments thereof, a T cell receptor (TCR) such as a tumor specific TCR and variants thereof, a chimeric antigen receptor (CAR), a chimeric switch receptor, a co-stimulatory molecule, a co-inhibitory molecule, an inhibitor of a co-inhibitory receptor or ligand, an agonist of a co-stimulatory receptor and ligand, a cytokine, chemokine, a cytokine receptor, a chemokine receptor, a soluble growth factor, a metabolic factor, a homing receptor, a safety switch (e.g., a suicide gene), or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anti-cancer immune response in a cell, or in a subject. In some aspects, the immunotherapeutic agent reduces the tumor burden in a subject.

The payload may be any immunotherapeutic agent used for cancer immunotherapy such as a T cell receptor (TCR), a chimeric agent receptor (CAR) such as CD19 CAR that targets any molecule of tumor cells, an antibody, an antigen binding domain or combination of antigen binding domains, a cytokine such as IL2, IL12, IL15 or IL15/IL15Ra fusion, an antagonist of an immune checkpoint, an agonist of co-stimulatory molecule, a chimeric switch receptor, a safety switch, a metabolic factor, a growth factor, a chemokine, a chemokine receptor, a homing receptor, or any agent that can induce an immune response. The SRE and payload may be operably linked through one or more linkers and the positions of components may vary within the effector module.

1. Protein of Interest

In some embodiments, payloads of the invention may be a natural protein in an organism genome, or variants, mutants, derivatives thereof. The natural protein may be from, for example, a mammalian organism, a bacterium, and a virus.

In one example, the payload may be a protein of interest, or a polypeptide from human genome.

2. Antibodies

In some embodiments, antibodies, fragments and variants thereof are payloads of the present invention. The antibody may be an intact antibody, an antibody light chain, antibody heavy chain, an antibody fragment, an antibody variant, or an antibody derivative.

In some embodiments, payloads of the invention may be an antibody or fragments thereof. Antibodies useful in this method include without limitation, any of those taught in co-owned U.S. Provisional Patent Application No. 62/320,864 filed Apr. 11, 2016, 62/466,596 filed Mar. 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety).

Antibody Fragments and Variants

In some embodiments, antibody fragments and variants may comprise antigen binding regions from intact antibodies. Examples of antibody fragments and variants may include, but are not limited to Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as single chain variable fragment (scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site. Also produced is a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-binding sites and is still capable of cross-linking with the antigen. Pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention may comprise one or more of these fragments.

For the purposes herein, an “antibody” may comprise a heavy and light variable domain as well as an Fc region. As used herein, the term “native antibody” usually refers to a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Genes encoding antibody heavy and light chains are known and segments making up each have been well characterized and described (Matsuda et al., The Journal of Experimental Medicine. 1998, 188(11): 2151-62 and Li et al., Blood, 2004, 103(12): 4602-4609; the content of each of which are herein incorporated by reference in their entirety). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

As used herein, the term “variable domain” refers to specific antibody domains found on both the antibody heavy and light chains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. Variable domains comprise hypervariable regions. As used herein, the term “hypervariable region” refers to a region within a variable domain comprising amino acid residues responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining regions (CDRs) that become part of the antigen-binding site of the antibody. As used herein, the term “CDR” refers to a region of an antibody comprising a structure that is complimentary to its target antigen or epitope. Other portions of the variable domain, not interacting with the antigen, are referred to as framework (FW) regions. The antigen-binding site (also known as the antigen combining site or paratope) comprises the amino acid residues necessary to interact with a particular antigen. The exact residues making up the antigen-binding site are typically elucidated by co-crystallography with bound antigen, however computational assessments based on comparisons with other antibodies can also be used (Strohl, W. R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia Pa. 2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety). Determining residues that make up CDRs may include the use of numbering schemes including, but not limited to, those taught by Kabat (Wu et al., JEM, 1970, 132(2):211-250 and Johnson et al., Nucleic Acids Res. 2000, 28(1): 214-218, the contents of each of which are herein incorporated by reference in their entirety), Chothia (Chothia and Lesk, J. Mol. Biol. 1987, 196, 901, Chothia et al., Nature, 1989, 342, 877, and A1-Lazikani et al., J. Mol. Biol. 1997, 273(4): 927-948, the contents of each of which are herein incorporated by reference in their entirety), Lefranc (Lefranc et al., Immunome Res. 2005, 1:3) and Honegger (Honegger and Pluckthun, J. Mol. Biol. 2001, 309(3): 657-70, the contents of which are herein incorporated by reference in their entirety).

VH and VL domains have three CDRs each. VL CDRs are referred to herein as CDR-L1, CDR-L2 and CDR-L3, in order of occurrence when moving from N- to C-terminus along the variable domain polypeptide. VH CDRs are referred to herein as CDR-H1, CDR-H2 and CDR-H3, in order of occurrence when moving from N- to C-terminus along the variable domain polypeptide. Each of CDRs has favored canonical structures with the exception of the CDR-H3, which comprises amino acid sequences that may be highly variable in sequence and length between antibodies resulting in a variety of three-dimensional structures in antigen-binding domains (Nikoloudis, et al., PeerJ. 2014, 2: e456). In some cases, CDR-H3s may be analyzed among a panel of related antibodies to assess antibody diversity. Various methods of determining CDR sequences are known in the art and may be applied to known antibody sequences (Strohl, W. R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia Pa. 2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety).

As used herein, the term “Fv” refers to an antibody fragment comprising the minimum fragment on an antibody needed to form a complete antigen-binding site. These regions consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. Fv fragments can be generated by proteolytic cleavage, but are largely unstable. Recombinant methods are known in the art for generating stable Fv fragments, typically through insertion of a flexible linker between the light chain variable domain and the heavy chain variable domain (to form a single chain Fv (scFv)) or through the introduction of a disulfide bridge between heavy and light chain variable domains (Strohl, W. R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia Pa. 2012. Ch. 3, p46-4′7, the contents of which are herein incorporated by reference in their entirety).

As used herein, the term “light chain” refers to a component of an antibody from any vertebrate species assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

As used herein, the term “single chain Fv” or “scFv” refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding. In some embodiments, scFvs are utilized in conjunction with phage display, yeast display or other display methods where they may be expressed in association with a surface member (e.g. phage coat protein) and used in the identification of high affinity peptides for a given antigen.

Using molecular genetics, two scFvs can be engineered in tandem into a single polypeptide, separated by a linker domain, called a “tandem scFv” (tascFv). Construction of a tascFv with genes for two different scFvs yields a “bispecific single-chain variable fragments” (bis-scFvs). Only two tascFvs have been developed clinically by commercial firms; both are bispecific agents in active early phase development by Micromet for oncologic indications, and are described as “Bispecific T-cell Engagers (BiTE).” Blinatumomab is an anti-CD19/anti-CD3 bispecific tascFv that potentiates T-cell responses to B-cell non-Hodgkin lymphoma in Phase 2. MT110 is an anti-EP-CAM/anti-CD3 bispecific tascFv that potentiates T-cell responses to solid tumors in Phase 1. Bispecific, tetravalent “TandAbs” are also being researched by Affimed (Nelson, A. L., MAbs., 2010, January-February; 2(1):77-83). maxibodies (bivalent scFv fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG may also be included.

As used herein, the term “bispecific antibody” refers to an antibody capable of binding two different antigens. Such antibodies typically comprise regions from at least two different antibodies. Bispecific antibodies may include any of those described in Riethmuller, G. Cancer Immunity. 2012, 12:12-18, Marvin et al., 2005. Acta Pharmacologica Sinica. 2005, 26(6): 649-658 and Schaefer et al., PNAS. 2011, 108(27):11187-11192, the contents of each of which are herein incorporated by reference in their entirety. In some aspects, bispecific antibodies may be trifunctional antibodies (3funct) and BiTE (bi-specific T cell engager).

As used herein, the term “diabody” refers to a small antibody fragment with two antigen-binding sites. Diabodies are functional bispecific single-chain antibodies (bscAb). Diabodies comprise a heavy chain variable domain VH connected to a light chain variable domain VL in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. (Hollinger, P. et al., “Diabodies”: Small bivalent and bispecific antibody fragments. PNAS, 1993. 90: 6444-6448); the contents of each of which are incorporated herein by reference in their entirety.

The term “intrabody” refers to a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies may be used to affect a multitude of cellular processes including, but not limited to intracellular trafficking, transcription, translation, metabolic processes, proliferative signaling and cell division. In some embodiments, methods of the present invention may include intrabody-based therapies. In some such embodiments, variable domain sequences and/or CDR sequences disclosed herein may be incorporated into one or more constructs for intrabody-based therapy.

As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

As used herein, the term “humanized antibody” refers to a chimeric antibody comprising a minimal portion from one or more non-human (e.g., murine) antibody source(s) with the remainder derived from one or more human immunoglobulin sources. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and/or capacity. In one embodiment, the antibody may be a humanized full-length antibody. As a non-limiting example, the antibody may have been humanized using the methods taught in US Patent Publication NO. US20130303399, the contents of which are herein incorporated by reference in its entirety.

As used herein, the term “antibody variant” refers to a modified antibody (in relation to a native or starting antibody) or a biomolecule resembling a native or starting antibody in structure and/or function (e.g., an antibody mimetic). Antibody variants may be altered in their amino acid sequence, composition or structure as compared to a native antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

In some embodiments, pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention may be antibody mimetics. As used herein, the term “antibody mimetic” refers to any molecule which mimics the function or effect of an antibody and which binds specifically and with high affinity to their molecular targets. In some embodiments, antibody mimetics may be monobodies, designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold (U.S. Pat. Nos. 6,673,901; 6,348,584). In some embodiments, antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, Centyrins, DARPINS™, Fynomers and Kunitz and domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.

In one embodiment, the antibody may comprise a modified Fc region. As a non-limiting example, the modified Fc region may be made by the methods or may be any of the regions described in US Patent Publication NO. US20150065690, the contents of which are herein incorporated by reference in its entirety.

In some embodiments, payloads of the invention may encode multispecific antibodies that bind more than one epitope. As used herein, the terms “multibody” or “multispecific antibody” refer to an antibody wherein two or more variable regions bind to different epitopes. The epitopes may be on the same or different targets. In one embodiment, the multispecific antibody may be generated and optimized by the methods described in International Patent Publication NO. WO2011109726 and US Patent Publication NO. US20150252119, the contents of which each of which are herein incorporated by reference in their entirety. These antibodies are able to bind to multiple antigens with high specificity and high affinity.

In certain embodiments, a multi-specific antibody is a “bispecific antibody” which recognizes two different epitopes on the same or different antigens. In one aspect, bispecific antibodies are capable of binding two different antigens. Such antibodies typically comprise antigen-binding regions from at least two different antibodies. For example, a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies, thus allowing the BsAb to bind to two different types of antigen. Bispecific antibody frameworks may include any of those described in Riethmuller, G., 2012. Cancer Immunity, 2012, 12:12-18; Marvin et al., Acta Pharmacologica Sinica. 2005, 26(6):649-658; and Schaefer et al., PNAS. 2011, 108(27): 11187-11192, the contents of each of which are herein incorporated by reference in their entirety. New generations of BsMAb, called “trifunctional bispecific” antibodies, have been developed. These consist of two heavy and two light chains, one each from two different antibodies, where the two Fab regions (the arms) are directed against two antigens, and the Fc region (the foot) comprises the two heavy chains and forms the third binding site.

In some embodiments, payloads may encode antibodies comprising a single antigen-binding domain. These molecules are extremely small, with molecular weights approximately one-tenth of those observed for full-sized mAbs. Further antibodies may include “nanobodies” derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas, which lack light chains (Nelson, A. L., MAbs.2010. January-February; 2(1):77-83).

In some embodiments, the antibody may be “miniaturized”. Among the best examples of mAb miniaturization are the small modular immunopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. These molecules, which can be monovalent or bivalent, are recombinant single-chain molecules containing one VL, one VH antigen-binding domain, and one or two constant “effector” domains, all connected by linker domains. Presumably, such a molecule might offer the advantages of increased tissue or tumor penetration claimed by fragments while retaining the immune effector functions conferred by constant domains. At least three “miniaturized” SMIPs have entered clinical development. TRU-015, an anti-CD20 SMIP developed in collaboration with Wyeth, is the most advanced project, having progressed to Phase 2 for rheumatoid arthritis (RA). Earlier attempts in systemic lupus erythrematosus (SLE) and B cell lymphomas were ultimately discontinued. Trubion and Facet Biotechnology are collaborating in the development of TRU-016, an anti-CD37 SMIP, for the treatment of CLL and other lymphoid neoplasias, a project that has reached Phase 2. Wyeth has licensed the anti-CD20 SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLE and possibly multiple sclerosis, although these projects remain in the earliest stages of clinical testing. (Nelson, A. L., MAbs, 2010. January-February; 2(1):77-83).

On example of miniaturized antibodies is called “unibody” in which the hinge region has been removed from IgG4 molecules. While IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with one another, deletion of the hinge region prevents heavy chain-heavy chain pairing entirely, leaving highly specific monovalent light/heavy heterodimers, while retaining the Fc region to ensure stability and half-life in vivo. This configuration may minimize the risk of immune activation or oncogenic growth, as IgG4 interacts poorly with FcRs and monovalent unibodies fail to promote intracellular signaling complex formation (see, e.g., Nelson, A. L., MAbs, 2010. January-February; 2(1):77-83).

In some embodiments, payloads of the invention may encode single-domain antibodies (sdAbs, or nanobodies) which are antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. In one aspect, a sdAb may be a “Camel Ig or “camelid VHH”. As used herein, the term “camel Ig” refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-No lte, et al, FASEB J., 2007, 21: 3490-3498). A “heavy chain antibody” or a “camelid antibody” refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al, J Immunol. Methods, 1999, 231: 25-38; International patent publication NOs.: WO1994/04678 and WO1994/025591; and U.S. Pat. No. 6,005,079). In another aspect, a sdAb may be a “immunoglobulin new antigen receptor” (IgNAR). As used herein, the term “immunoglobulin new antigen receptor” refers to class of antibodies from the shark immune repertoire that consist of homodimers of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains. IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and are highly stable and possess efficient binding characteristics. The inherent stability can be attributed to both (i) the underlying Ig scaffold, which presents a considerable number of charged and hydrophilic surface exposed residues compared to the conventional antibody VH and VL domains found in murine antibodies; and (ii) stabilizing structural features in the complementary determining region (CDR) loops including inter-loop disulphide bridges, and patterns of intra-loop hydrogen bonds.

In some embodiments, payloads of the invention may encode intrabodies. Intrabodies are a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies are expressed and function intracellularly, and may be used to affect a multitude of cellular processes including, but not limited to intracellular trafficking, transcription, translation, metabolic processes, proliferative signaling and cell division. In some embodiments, methods described herein include intrabody-based therapies. In some such embodiments, variable domain sequences and/or CDR sequences disclosed herein are incorporated into one or more constructs for intrabody-based therapy. For example, intrabodies may target one or more glycated intracellular proteins or may modulate the interaction between one or more glycated intracellular proteins and an alternative protein.

The intracellular expression of intrabodies in different compartments of mammalian cells allows blocking or modulation of the function of endogenous molecules (Biocca, et al., EMBO J. 1990, 9: 101-108; Colby et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101: 17616-17621). Intrabodies can alter protein folding, protein-protein, protein-DNA, protein-RNA interactions and protein modification. They can induce a phenotypic knockout and work as neutralizing agents by direct binding to the target antigen, by diverting its intracellular trafficking or by inhibiting its association with binding partners. With high specificity and affinity to target antigens, intrabodies have advantages to block certain binding interactions of a particular target molecule, while sparing others.

Sequences from donor antibodies may be used to develop intrabodies. Intrabodies are often recombinantly expressed as single domain fragments such as isolated VH and VL domains or as a single chain variable fragment (scFv) antibody within the cell. For example, intrabodies are often expressed as a single polypeptide to form a single chain antibody comprising the variable domains of the heavy and light chains joined by a flexible linker polypeptide. Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Single chain intrabodies are often expressed from a recombinant nucleic acid molecule and engineered to be retained intracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum, or periplasm). Intrabodies may be produced using methods known in the art, such as those disclosed and reviewed in: (Marasco et al., PNAS, 1993, 90: 7889-7893; Chen et al., Hum. Gene Ther. 1994, 5:595-601; Chen et al., 1994, PNAS, 91: 5932-5936; Maciejewski et al., 1995, Nature Med., 1: 667-673; Marasco, 1995, Immunotech, 1: 1-19; Mhashilkar, et al., 1995, EMBO J. 14: 1542-51; Chen et al., 1996, Hum. Gene Therap., 7: 1515-1525; Marasco, Gene Ther. 4:11-15, 1997; Rondon and Marasco, 1997, Annu. Rev. Microbiol. 51:257-283; Cohen, et al., 1998, Oncogene 17:2445-56; Proba et al., 1998, J. Mol. Biol. 275:245-253; Cohen et al., 1998, Oncogene 17:2445-2456; Hassanzadeh, et al., 1998, FEBS Lett. 437:81-6; Richardson et al., 1998, Gene Ther. 5:635-44; Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291:1129-1134; Wirtz and Steipe, 1999, Protein Sci. 8:2245-2250; Zhu et al., 1999, J. Immunol. Methods 231:207-222; Arafat et al., 2000, Cancer Gene Ther. 7:1250-6; der Maur et al., 2002, J. Biol. Chem. 277:45075-85; Mhashilkar et al., 2002, Gene Ther. 9:307-19; and Wheeler et al., 2003, FASEB J. 17: 1733-5; and references cited therein).

In some embodiments, payloads of the invention may encode biosynthetic antibodies as described in U.S. Pat. No. 5,091,513, the contents of which are herein incorporated by reference in their entirety. Such antibody may include one or more sequences of amino acids constituting a region which behaves as a biosynthetic antibody binding site (BABS). The sites comprise 1) non-covalently associated or disulfide bonded synthetic VH and VL dimers, 2) VH-VL or VL-VH single chains wherein the VH and VL are attached by a polypeptide linker, or 3) individuals VH or VL domains. The binding domains comprise linked CDR and FR regions, which may be derived from separate immunoglobulins. The biosynthetic antibodies may also include other polypeptide sequences which function, e.g., as an enzyme, toxin, binding site, or site of attachment to an immobilization media or radioactive atom. Methods are disclosed for producing the biosynthetic antibodies, for designing BABS having any specificity that can be elicited by in vivo generation of antibody, and for producing analogs thereof.

In some embodiments, payloads may encode antibodies with antibody acceptor frameworks taught in U.S. Pat. No. 8,399,625. Such antibody acceptor frameworks may be particularly well suited accepting CDRs from an antibody of interest.

In one embodiment, the antibody may be a conditionally active biologic protein. An antibody may be used to generate a conditionally active biologic protein which are reversibly or irreversibly inactivated at the wild type normal physiological conditions as well as to such conditionally active biologic proteins and uses of such conditional active biologic proteins are provided. Such methods and conditionally active proteins are taught in, for example, International Publication No. WO2015175375 and WO2016036916 and US Patent Publication No. US20140378660, the contents of each of which are incorporated herein by reference in their entirety.

Antibody Preparations

The preparation of antibodies, whether monoclonal or polyclonal, is known in the art. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999 and “Therapeutic Antibody Engineering: Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry” Woodhead Publishing, 2012.

The antibodies and fragments and variants thereof as described herein can be produced using recombinant polynucleotides. In one embodiment, the polynucleotides have a modular design to encode at least one of the antibodies, fragments or variants thereof. As a non-limiting example, the polynucleotide construct may encode any of the following designs: (1) the heavy chain of an antibody, (2) the light chain of an antibody, (3) the heavy and light chain of the antibody, (4) the heavy chain and light chain separated by a linker, (5) the VH1, CH1, CH2, CH3 domains, a linker and the light chain or (6) the VH1, CH1, CH2, CH3 domains, VL region, and the light chain. Any of these designs may also comprise optional linkers between any domain and/or region. The polynucleotides of the present invention may be engineered to produce any standard class of immunoglobulins using an antibody described herein or any of its component parts as a starting molecule.

Antibodies Used for Immunotherapy

In some embodiments, payloads of the present invention may be antibodies, fragments and variants thereof which are specific to tumor specific antigens (TSAs) and tumor associated antigens (TAAs). Antibodies circulate throughout the body until they find and attach to the TSA/TAA. Once attached, they recruit other parts of the immune system, increasing ADCC (antibody dependent cell-mediated cytotoxicity) and ADCP (antibody dependent cell-mediated phagocytosis) to destroy tumor cells. As used herein, the term “tumor specific antigen (TSA)” means an antigenic substance produced in tumor cells, which can trigger an anti-tumor immune response in a host organism. In one embodiment, a TSA may be a tumor neoantigen. The tumor antigen specific antibody mediates complement-dependent cytotoxic response against tumor cells expressing the same antigen.

Of particular interest is a TSA that is a breast cancer antigen, an ovarian cancer antigen, a prostate cancer antigen, a cervical cancer antigen, a pancreatic carcinoma antigen, a lung cancer antigen, a bladder cancer antigen, a colon cancer antigen, a testicular cancer antigen, a glioblastoma cancer antigen, an antigen associated with a B cell malignancy, an antigen associated with multiple myeloma, an antigen associated with non-Hodgkins lymphoma, or an antigen associated with chronic lymphocytic leukemia.

Suitable antibodies which can immunoactively bind to a TSA may include, but are not limited to, those specific to 5T4, 707-AP, A33, AFP (α-fetoprotein), AKAP-4 (A kinase anchor protein 4), ALK, α5β1-integrin, androgen receptor, annexin II, alpha-actinin-4, ART-4, B1, B7H3, B7H4, BAGE (B melanoma antigen), BCMA, BCR-ABL fusion protein, beta-catenin, BKT-antigen, BTAA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calretinin, CAIX (carbonic anhydrase), CAMEL (cytotoxic T-lymphocyte recognized antigen on melanoma), CAM43, CAP-1, Caspase-8/m, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD25, CD27/m, CD28, CD30, CD33, CD34, CD36, CD38, CD40/CD154, CD41, CD44v6, CD44v7/8, CD45, CD49f, CD56, CD68\KP1, CD74, CD79a/CD79b, CD103, CD123, CD133, CD138, CD171, cdc27/m, CDK4 (cyclin dependent kinase 4), CDKN2A, CD S, CEA (carcinoembryonic antigen), CEACAM5, CEACAM6, chromogranin, c-Met, c-Myc, coa-1, CSAp, CT7, CT10, cyclophilin B, cyclin B1, cytoplasmic tyrosine kinases, cytokeratin, DAM-10, DAM-6, dek-can fusion protein, desmin, DEPDC1 (DEP domain containing 1), E2A-PRL, EBNA, EGF-R (epidermal growth factor receptor), EGP-1 (epithelial glycoprotein −1) (TROP-2), EGP-2, EGP-40, EGFR (epidermal growth factor receptor), EGFRvIII, EF-2, ELF2M, EMMPRIN, EpCAM (epithelial cell adhesion molecule), EphA2, Epstein Barr virus antigens, Erb (ErbB1; ErbB3; ErbB4), ETA (epithelial tumor antigen), ETV6-AML1 fusion protein, FAP (fibroblast activation protein), FBP (folate-binding protein), FGF-5, folate receptor a, FOS related antigen 1, fucosyl GM1, G250, GAGE (GAGE-1; GAGE-2), galactin, GD2 (ganglioside), GD3, GFAP (glial fibrillary acidic protein), GM2 (oncofetal antigen-immunogenic-1; OFA-I-1), GnT-V, Gp100, H4-RET, HAGE (helicase antigen), HER-2/neu, HIFs (hypoxia inducible factors), HIF-1α, HIF-2α, HLA-A2, HLA-A*0201-R170I, HLA-A11, HMWMAA, Hom/Mel-40, HSP70-2M (Heat shock protein 70), HST-2, HTgp-175, hTERT (or hTRT), human papillomavirus-E6/human papillomavirus-E7 and E6, iCE (immune-capture EIA), IGF-1R, IGH-IGK, IL2R, IL5, ILK (integrin-linked kinase), IMP3 (insulin-like growth factor II mRNA-binding protein 3), IRF4 (interferon regulatory factor 4), KDR (kinase insert domain receptor), KIAA0205, KRAB-zinc finger protein (KID)-3; KID31, KSA (17-1A), K-ras, LAGE, LCK, LDLR/FUT (LDLR-fucosyltransferaseAS fusion protein), LeY (Lewis Y), MAD-CT-1, MAGE (tyrosinase, melanoma-associated antigen) (MAGE-1; MAGE-3), melan-A tumor antigen (MART), MART-2/Ski, MC1R (melanocortin 1 receptor), MDM2, mesothelin, MPHOSPH1, MSA (muscle-specific actin), mTOR (mammalian targets of rapamycin), MUC-1, MUC-2, MUM-1 (melanoma associated antigen (mutated) 1), MUM-2, MUM-3, Myosin/m, MYL-RAR, NA88-A, N-acetylglucosaminyltransferase, neo-PAP, NF-KB (nuclear factor-kappa B), neurofilament, NSE (neuron-specific enolase), Notch receptors, NuMa, N-Ras, NY-BR-1, NY-CO-1, NY-ESO-1, Oncostatin M, OS-9, OY-TES1, p53 mutants, p190 minor bcr-ab1, p15(58), p185erbB2, p180erbB-3, PAGE (prostate associated gene), PAP (prostatic acid phosphatase), PAX3, PAX5, PDGFR (platelet derived growth factor receptor), cytochrome P450 involved in piperidine and pyrrolidine utilization (PIPA), Pm1-RAR alpha fusion protein, PR-3 (proteinase 3), PSA (prostate specific antigen), PSM, PSMA (Prostate stem cell antigen), PRAME (preferentially expressed antigen of melanoma), PTPRK, RAGE (renal tumor antigen), Raf (A-Raf, B-Raf and C-Raf), Ras, receptor tyrosine kinases, RCAS1, RGSS, ROR1 (receptor tyrosine kinase-like orphan receptor 1), RU1, RU2, SAGE, SART-1, SART-3, SCP-1, SDCCAG16, SP-17 (sperm protein 17), src-family, SSX (synovial sarcoma X breakpoint)-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, STAT-3, STAT-5, STAT-6, STEAD, STn, survivin, syk-ZAP70, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TACSTD1 (tumor associated calcium signal transducer 1), TACSTD2, TAG-72-4, TAGE, TARP (T cell receptor gamma alternate reading frame protein), TEL/AML1 fusion protein, TEM1, TEM8 (endosialin or CD248), TGFβ, TIE2, TLP, TMPRSS2 ETS fusion gene, TNF-receptor (TNF-α receptor, TNF-β receptor; or TNF-γ receptor), transferrin receptor, TPS, TRP-1 (tyrosine related protein 1), TRP-2, TRP-2/INT2, TSP-180, VEGF receptor, WNT, WT-1 (Wilm's tumor antigen) and XAGE.

In one embodiment, the payload of the present invention may be an anti-CD47 antibody. CD47 is a ubiquitously expressed immunoregulatory protein that prevents phagocytic removal of healthy cells by the immune system. CD47 is expressed on the surface of many types of cancer cells, thereby disrupting anti-cancer immune responses. CD 47 is also involved in various other important cellular processes, such as angiogenesis, cancer cell death and regulation of T-cell immunity. Anti-CD47 antibodies in several pre-clinical studies have shown therapeutic benefit in solid cancers and most notably B-cell malignancies.

In one embodiment, the payload of the present invention may be an anti-CD22 antibody. As a non-limiting example, the anti-CD22 antibody is any of the antibodies, fragments or variants thereof described in US Patent Publication No. US20150086562. The anti-CD22 antibody may comprise a heavy chain variable region having the amino acid sequences of SEQ ID NO: 49-64 in US20150086562, and/or a light chain variable region having the amino acid sequence of SEQ ID NO: 17-32 in US20150086562; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, payloads of the present invention may be antibodies, fragments and variants thereof which can specifically block an immunoinhibitory signal. These blocking antibodies (also referred to as antagonists) bind to co-inhibitory receptors, therefore blocking their signal transduction. As non-limiting examples, the blocking antibodies may be specific to CTLA-4, PD-1, and PD-L1/L2. In one embodiment, the anti-CTLA-4 antibody is Ipilimumab. In another embodiment, the anti-PD-1 antibody is Nivolumab. Antibodies that bind to PD-L1 and enhance T cell immune response may include antibodies taught in US patent publication NO.: 2016/0108123; the contents of which are incorporated by reference herein in their entirety. Other inhibitory immunomodulatory targets may include B7-H3, which can increase cancer cell metabolism such as glucose uptakes and lactate production (Lim et al., Cancer Res., 2016, 76(8): 1-12). Antibodies that block B7-H3 are disclosed in U.S. Pat. No. 9,150,656; the contents of which are incorporated by reference herein in their entirety.

In one aspect, payloads of the present invention may be antagonistic antibodies specific to VSIG8 (v-set and immunoglobulin domain containing 8) comprising the amino acid sequences of SEQ ID NOs: 1, 2 and 3 in US patent publication NO.: US2016/0159927; the contents of which are incorporated by reference herein in their entirety. Antagonistic antibodies may also include a chimeric IL2 receptor (CD25) antibody (Basiliximab) (US Patent Publication NO.: 20080171017; the contents of which are incorporated herein by reference in their entirety), and antagonizing antibodies which bind to human TIM-3 (US patent publication NO.: US2015/0218274; the contents of which are incorporated herein by reference in their entirety), BTLA, VISTA and LAG-3 (See, e.g., US patent publication NO.: US2015/0259420; the contents of which are incorporated herein by reference in their entirety).

In one aspect, the payload of the present invention may be an anti-CSF-IR antibody, which is characterized in binding to the dimerization domains D4 to D5 of the extracellular domain of human CSF-IR. This antibody inhibitor can inhibit cell proliferation and survival in CSF-IR ligand-dependent and CSF-1 ligand-independent CSF-IR expressing tumor cells, monocytes and infiltrating macrophages (See, e.g., International Patent Publication NO.: WO2013/132044; the contents of which are incorporated herein by reference in their entirety).

In another aspect, the payload of the present invention may be an antagonistic antibody against CXCL12. The anti-CXCL12 antibody blocks the interaction of CXCL12 with its receptor CXCR4, thereby inhibiting CXCR4 signaling. The CXCR4 signaling inhibitor increases the proximity or the frequency of T-cells among cancer cells in the tumor tissue (See, International Patent Publication NO.: WO 2015/019284; the contents of which are incorporated by reference herein in their entirety).

In some embodiments, payloads of the present invention may be agonistic antibodies, fragments and variants thereof, which trigger immune responses, including antibodies specific to co-stimulatory molecules, including but not limited to 4-1BB (CD137), OX40 (CD134), CD40, GITR and CD27.

In one embodiment, the payload of the present invention may be an agonistic CD40 antibody. In another embodiment, the agonistic antibody specific to 4-1BB (CD137) may be Uremab, a fully human IgG4 monoclonal antibody which specifically binds to and activates 4-1BB (CD137) expressing immune cells, stimulating an immune response, in particular a cytotoxic T cell response, against tumor cells; or Utomilumab, a fully human IgG2 monoclonal antibody; or anti-CD137 antibody described in International Patent Publication NO.: WO2006/088447; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the payload of the present invention may be an antagonistic antibody against Amphiregulin. Amphiregulin (AREG) is an EGF-like growth factor which binds to the EGFR receptor and enhances T regulatory cell function. AREG is produced in a phenotypically and functionally distinct subtype of CD4^(+ regulatory T cells (Tregs) which have a distinct T cell receptor (TCR) repertoire and express the IL)33R. AREG promotes immune suppression in the tumor environment. The anti-Amphiregulin antibody may comprise a heavy chain variable region having the amino acid sequences of SEQ ID NO.: 2,4, and 12 in U.S. Pat. No. 7,223,393, and/or a light chain variable region having the amino acid sequence of SEQ ID NO.: 3, 5, and 14 in U.S. Pat. No. 7,223,393; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, antibodies specific to co-inhibitory molecules and co-stimulatory molecules may be secreted scFv antibodies.

In some embodiments, antibody payloads of the present invention may be T-cell bispecific antibodies (e.g. T cell-engaging BiTE™ antibodies CDS-CD 19, CD3-EpCam, and CD3-EGFR). Other bispecific antibodies used for immunotherapy may also be included as payloads of the present invention, for example, bispecific anti-TNF-α and anti-IL6 antibody (EP3062818), bispecific antibodies to an immune cell antigen and TAG-72 (WO2016/089610), anti-ovarian and D3 bispecific antibodies in U.S. Pat. No. 7,262,276; bispecific antibodies against CD133 and CD3 in WO2014/128185; bispecific antibodies against CTLA-4 and PD-1 discussed in US2016/0145355, bispecific antibodies against CD3 and CD19 disclosed in WO2015/006749, and US. Pat. NOs. 7, 635,472; 7, 112, 324; bispecific antibodies against Her2 and CD3 in US2014/0170149; bispecific antibodies against CD19 and CD16 in US2005/0089519; the contents of each of which are incorporated by reference herein in their entirety.

In some embodiments, antibodies with decreased affinity may be selected over antibodies with high affinity for the same antigen. Such low affinity antibodies are more effective in discriminating tumors which express high levels of the antigen and normal tissues that express the same antigen at lower levels, while maintaining similar antitumor response.

In some embodiments, the targeting moieties of the present invention may include variable heavy chain and variable light chain comprising the amino acid sequences selected from those in Table 10.

TABLE 10 Variable Heavy and Light Chain Sequences Antibody SEQ Target chain ID NO Source 5T4 VH 539 SEQ ID NO. 2 in WO2016022939 5T4 VH 540 SEQ ID NO. 4 in WO2016022939 AGR2 VH 541 SEQ ID NO. 10 in WO2016040321 AGR2 VH 542 SEQ ID NO. 18 in WO2016040321 ALK VH 543 SEQ ID NO. 11 in WO2015069922 ALK VH 544 SEQ ID NO. 13 in WO2015069922 ALK VH 545 SEQ ID NO. 15 in WO2015069922 ALK VH 546 SEQ ID NO. 7 in WO2015069922 ALK VH 547 SEQ ID NO. 9 in WO2015069922 ALK VH 548 SEQ ID NO: 1 in US20160280798A1 ALK VH 549 SEQ ID NO: 11 in US20160280798A1 ALK VH 550 SEQ ID NO: 13 in US20160280798A1 ALK VH 551 SEQ ID NO: 15 in US20160280798A1 ALK VH 552 SEQ ID NO: 3 in US20160280798A1 ALK VH 553 SEQ ID NO: 5 in US20160280798A1 ALK VH 554 SEQ ID NO: 7 in US20160280798A1 ALK VH 555 SEQ ID NO: 9 in US20160280798A1 ALK VH 556 SEQ ID NO. 1 in WO2015069922 ALK VH 557 SEQ ID NO. 3 in WO2015069922 ALK VH 558 SEQ ID NO. 5 in WO2015069922 AMC VH 559 SEQ ID NO. 17 in WO2016161390 AMC VH 560 SEQ ID NO. 18 in WO2016161390 AMC VH 561 SEQ ID NO. 19 in WO2016161390 AMC VH 562 SEQ ID NO. 20 in WO2016161390 AMC VH 563 SEQ ID NO. 21 in WO2016161390 AMC VH 564 SEQ ID NO. 22 in WO2016161390 AMC VH 565 SEQ ID NO. 23 in WO2016161390 AMC VH 566 SEQ ID NO. 24 in WO2016161390 AMC VH 567 SEQ ID NO. 25 in WO2016161390 AMC VH 568 SEQ ID NO. 26 in WO2016161390 ANG2 VH 569 SEQ ID NO. 1 in WO2015091655 ANG2 VH 570 SEQ ID NO. 3 in WO2015091655 APCDD1 VH 571 SEQ ID NO: 10 in WO2012019061 APCDD1 VH 572 SEQ ID NO: 102 in WO2012019061 APCDD1 VH 573 SEQ ID NO: 106 in WO2012019061 APCDD1 VH 574 SEQ ID NO: 110 in WO2012019061 APCDD1 VH 575 SEQ ID NO: 114 in WO2012019061 APCDD1 VH 576 SEQ ID NO: 118 in WO2012019061 APCDD1 VH 577 SEQ ID NO: 122 in WO2012019061 APCDD1 VH 578 SEQ ID NO: 126 in WO2012019061 APCDD1 VH 579 SEQ ID NO: 130 in WO2012019061 APCDD1 VH 580 SEQ ID NO: 134 in WO2012019061 APCDD1 VH 581 SEQ ID NO: 14 in WO2012019061 APCDD1 VH 582 SEQ ID NO: 6 in WO2012019061 APCDD1 VH 583 SEQ ID NO: 98 in WO2012019061 APRIL VH 584 SEQ ID NO. 12 in US20160264674 APRIL VH 585 SEQ ID NO. 14 in US20160264674 APRIL VH 586 SEQ ID NO. 16 in US20160264674 APRIL VH 587 SEQ ID NO. 18 in US20160264674 APRIL VH 588 SEQ ID NO. 3 in US20160264674 APRIL VH 589 SEQ ID NO. 32 in US20160264674 APRIL VH 590 SEQ ID NO. 34 in US20160264674 APRIL VH 591 SEQ ID NO. 36 in US20160264674 APRIL VH 592 SEQ ID NO. 38 in US20160264674 APRIL VH 593 SEQ ID NO. 40 in US20160264674 APRIL VH 594 SEQ ID NO. 42 in US20160264674 APRIL VH 595 SEQ ID NO. 44 in US20160264674 APRIL VH 596 SEQ ID NO. 46 in US20160264674 APRIL VH 597 SEQ ID NO. 48 in US20160264674 APRIL VH 598 SEQ ID NO. 52 in US20160264674 AXL VH 599 SEQ ID NO. 21 in WO2016097370 AXL VH 600 SEQ ID NO. 3 in WO2016097370 AXL VH 601 SEQ ID NO. 45 in WO2016097370 B2MG VH 602 SEQIDNO: 28 in WO2016126213A1 B7H1 VH 603 SEQ ID NO: 12 in US20130034559 B7H1 VH 604 SEQ ID NO: 32 in US20130034559 B7H1 VH 605 SEQ ID NO: 42 in US20130034559 B7H1 VH 606 SEQ ID NO: 52 in US20130034559 B7H1 VH 607 SEQ ID NO: 72 in US20130034559 B7H1 VH 608 SEQ ID NO: 2 in US20130034559 B7H1 VH 609 SEQ ID NO: 62 in US20130034559 B7H3 VH 610 SEQ ID NO. 10 in WO2016033225 B7H3 VH 611 SEQ ID NO. 11 in WO2016033225 B7H3 VH 612 SEQ ID NO. 12 in WO2016033225 B7H3 VH 613 SEQ ID NO. 13 in WO2016033225 B7H3 VH 614 SEQ ID NO. 14 in WO2016033225 B7H3 VH 615 SEQ ID NO. 15 in WO2016033225 B7H3 VH 616 SEQ ID NO. 16 in WO2016033225 B7H3 VH 617 SEQ ID NO. 9 in WO2016033225 B7H3(CD276) VH 618 SEQ ID NO. 17 in WO2016044383 B7H3(CD276) VH 619 SEQ ID NO. 26 in WO2016044383 B7H3(CD276) VH 620 SEQ ID NO. 7 in WO2016044383 B7H4 VH 621 SEQ ID NO. 100 in US20160159910 B7H4 VH 622 SEQ ID NO. 101 in US20160159910 B7H4 VH 623 SEQ ID NO. 102 in US20160159910 B7H4 VH 624 SEQ ID NO. 103 in US20160159910 B7H4 VH 625 SEQ ID NO. 107 in US20160159910 B7H4 VH 626 SEQ ID NO. 108 in US20160159910 B7H4 VH 627 SEQ ID NO. 109 in US20160159910 B7H4 VH 628 SEQ ID NO. 110 in US20160159910 B7H4 VH 629 SEQ ID NO. 111 in US20160159910 B7H4 VH 630 SEQ ID NO. 112 in US20160159910 B7H4 VH 631 SEQ ID NO. 113 in US20160159910 B7H4 VH 632 SEQ ID NO. 114 in US20160159910 B7H4 VH 633 SEQ ID NO. 12 in US20160159910 B7H4 VH 634 SEQ ID NO. 127 in US20160159910 B7H4 VH 635 SEQ ID NO. 13 in WO2016160620 B7H4 VH 636 SEQ ID NO. 130 in US20160159910 B7H4 VH 637 SEQ ID NO. 131 in US20160159910 B7H4 VH 638 SEQ ID NO. 132 in US20160159910 B7H4 VH 639 SEQ ID NO. 133 in US20160159910 B7H4 VH 640 SEQ ID NO. 137 in US20160159910 B7H4 VH 641 SEQ ID NO. 2 in US20160159910 B7H4 VH 642 SEQ ID NO. 20 in US20160159910 B7H4 VH 643 SEQ ID NO. 28 in US20160159910 B7H4 VH 644 SEQ ID NO. 36 in US20160159910 B7H4 VH 645 SEQ ID NO. 37 in US20160159910 B7H4 VH 646 SEQ ID NO. 38 in US20160159910 B7H4 VH 647 SEQ ID NO. 4 in US20160159910 B7H4 VH 648 SEQ ID NO. 56 in US20160159910 B7H4 VH 649 SEQ ID NO. 99 in US20160159910 B7H4 VH 650 SEQ ID NO. 144 in US20160159910 B7H4 VH 651 SEQ ID NO. 15 in WO2016160620 B7H4 VH 652 SEQ ID NO. 17 in WO2016160620 BAT1 VH 653 SEQ ID NO. 5 in WO2013014668 BAT1 VH 654 SEQ ID NO. 6 in WO2013014668 BAT1 VH 655 SEQ ID NO. 7 in WO2013014668 BAT1 VH 656 SEQ ID NO. 8 in WO2013014668 BAT1 VH 657 SEQ ID NO. 9 in WO2013014668 BCMA VH 658 SEQ ID NO: 26 in WO2016168773A3 BCMA VH 659 SEQ ID NO: 142 in WO2016168595A1 BCMA VH 660 SEQ ID NO: 148 in WO2016168595A1 BCMA VH 661 SEQ ID NO: 154 in WO2016168595A1 BCMA VH 662 SEQ ID NO: 160 in WO2016168595A1 BCMA VH 663 SEQ ID NO: 166 in WO2016168595A1 BCMA VH 664 SEQ ID NO: 172 in WO2016168595A1 BCMA VH 665 SEQ ID NO: 178 in WO2016168595A1 BCMA VH 666 SEQ ID NO: 184 in WO2016168595A1 BCMA VH 667 SEQ ID NO: 190 in WO2016168595A1 BCMA VH 668 SEQ ID NO: 196 in WO2016168595A1 BCMA VH 669 SEQ ID NO: 202 in WO2016168595A1 BCMA VH 670 SEQ ID NO: 208 in WO2016168595A1 BCMA VH 671 SEQ ID NO: 214 in WO2016168595A1 BCMA VH 672 SEQ ID NO: 220 in WO2016168595A1 BCMA VH 673 SEQ ID NO: 226 in WO2016168595A1 BCMA VH 674 SEQ ID NO: 232 in WO2016168595A1 BCMA VH 675 SEQ ID NO: 238 in WO2016168595A1 BCMA VH 676 SEQ ID NO: 244 in WO2016168595A1 BCMA VH 677 SEQ ID NO: 250 in WO2016168595A1 BCMA VH 678 SEQ ID NO: 256 in WO2016168595A1 BCMA VH 679 SEQ ID NO: 262 in WO2016168595A1 BCMA VH 680 SEQ ID NO: 268 in WO2016168595A1 BCMA VH 681 SEQ ID NO: 274 in WO2016168595A1 BCMA VH 682 SEQ ID NO: 280 in WO2016168595A1 BCMA VH 683 SEQ ID NO: 286 in WO2016168595A1 BCMA VH 684 SEQ ID NO: 292 in WO2016168595A1 BCMA VH 685 SEQ ID NO: 298 in WO2016168595A1 BCMA VH 686 SEQ ID NO: 304 in WO2016168595A1 BCMA VH 687 SEQ ID NO: 310 in WO2016168595A1 BCMA VH 688 SEQ ID NO: 316 in WO2016168595A1 BCMA VH 689 SEQ ID NO: 322 in WO2016168595A1 BCMA VH 690 SEQ ID NO: 328 in WO2016168595A1 BCMA VH 691 SEQ ID NO: 334 in WO2016168595A1 BCMA VH 692 SEQ ID NO: 340 in WO2016168595A1 BCMA VH 693 SEQ ID NO: 346 in WO2016168595A1 BCMA VH 694 SEQ ID NO: 352 in WO2016168595A1 BCMA VH 695 SEQ ID NO: 8 in WO2016094304A3 BCMA VH 696 SEQ ID NO. 171 WO2016014565 BCMA VH 697 SEQ ID NO. 172 WO2016014565 BCMA VH 698 SEQ ID NO. 173 WO2016014565 BCMA VH 699 SEQ ID NO. 174 WO2016014565 BCMA VH 700 SEQ ID NO. 175 WO2016014565 BCMA VH 701 SEQ ID NO. 176 WO2016014565 BCMA VH 702 SEQ ID NO. 177 WO2016014565 BCMA VH 703 SEQ ID NO. 178 WO2016014565 BCMA VH 704 SEQ ID NO. 179 WO2016014565 BCMA VH 705 SEQ ID NO. 180 WO2016014565 BCMA VH 706 SEQ ID NO. 181 WO2016014565 BCMA VH 707 SEQ ID NO. 182 WO2016014565 BCMA VH 708 SEQ ID NO. 183 WO2016014565 BCMA VH 709 SEQ ID NO. 184 WO2016014565 BCMA VH 710 SEQ ID NO. 185 WO2016014565 BCMA VH 711 SEQ ID NO. 186 WO2016014565 BCMA VH 712 SEQ ID NO. 187 WO2016014565 BCMA VH 713 SEQ ID NO. 190 WO2016014565 BCMA VH 714 SEQ ID NO. 255 WO2016014565 BCMA VH 715 SEQ ID NO. 257 WO2016014565 BCMA VH 716 SEQ ID NO. 258 WO2016014565 BCMA VH 717 SEQ ID NO. 69WO2016014565 BCMA VH 718 SEQ ID NO. 70WO2016014565 BCMA VH 719 SEQ ID NO. 71 WO2016014565 BCMA VH 720 SEQ ID NO. 72 WO2016014565 BCMA VH 721 SEQ ID NO. 73 WO2016014565 BCMA VH 722 SEQ ID NO. 74 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WO2016090320 BCMA VH 747 SEQ ID NO. 33 in WO2016090320 BCMA VH 748 SEQ ID NO. 37 in WO2016090320 BCMA VH 749 SEQ ID NO. 41 in WO2016090320 BCMA VH 750 SEQ ID NO. 45 in WO2016090320 BCMA VH 751 SEQ ID NO. 49 in WO2016090320 BCMA VH 752 SEQ ID NO. 5 in WO2016090320 BCMA VH 753 SEQ ID NO. 53 in WO2016090320 BCMA VH 754 SEQ ID NO. 57 in WO2016090320 BCMA VH 755 SEQ ID NO. 61 in WO2016090320 BCMA VH 756 SEQ ID NO. 65 in WO2016090320 BCMA VH 757 SEQ ID NO. 9 in WO2016090320 BCMA VH 758 SEQ ID NO. 95 in WO2016120216 BCMA VH 759 SEQ ID NO. 97 in WO2016120216 BCMA VH 760 SEQ ID NO. 99 in WO2016120216 BCMA VH 761 SEQ ID NO: 15 in WO2016168766A1 BMPR1A VH 762 SEQ ID NO. 12 in WO2011116212 CA19.9 VH 763 SEQ ID NO: 117 in US20160333114A1 Campath1 VH 764 SEQ ID NO: 34 in US20160333114A1 CD105 VH 765 SEQ ID NO. 13 in WO2014039682 CD105 VH 766 SEQ ID NO. 14 in WO2014039682 CD105 VH 767 SEQ ID NO. 16 in WO2014039682 CD123 VH 768 SEQ ID NO. 11 in WO2015140268A1 CD123 VH 769 SEQ ID NO. 113 in 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SEQ ID NO. 35 in US20140105914 CTLA4 VH 1340 SEQ ID NO. 4 in U.S. Pat. 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DR5(Drozitumab) VH 1362 SEQ ID NO. 58 in WO2016122701 DR5(Tigatuzumab) VH 1363 SEQ ID NO. 74 in WO2016122701 E7MC VH 1364 SEQ ID NO: 15 in WO2016182957A1 E7MC VH 1365 SEQ ID NO: 16 in WO2016182957A1 E7MC VH 1366 SEQ ID NO: 17 in WO2016182957A1 E7MC VH 1367 SEQ ID NO: 18 in WO2016182957A1 E7MC VH 1368 SEQ ID NO: 19 in WO2016182957A1 E7MC VH 1369 SEQ ID NO: 20 in WO2016182957A1 E7MC VH 1370 SEQ ID NO: 21 in WO2016182957A1 E7MC VH 1371 SEQ ID NO: 22 in WO2016182957A1 E7MC VH 1372 SEQ ID NO: 23 in WO2016182957A1 E7MC VH 1373 SEQ ID NO: 233 in WO2016182957A1 E7MC VH 1374 SEQ ID NO: 234 in WO2016182957A1 E7MC VH 1375 SEQ ID NO: 235 in WO2016182957A1 E7MC VH 1376 SEQ ID NO: 236 in WO2016182957A1 E7MC VH 1377 SEQ ID NO: 237 in WO2016182957A1 E7MC VH 1378 SEQ ID NO: 24 in WO2016182957A1 E7MC VH 1379 SEQ ID NO: 25 in WO2016182957A1 E7MC VH 1380 SEQ ID NO: 26 in WO2016182957A1 E7MC VH 1381 SEQ ID NO: 27 in WO2016182957A1 E7MC VH 1382 SEQ ID NO: 28 in WO2016182957A1 E7MC VH 1383 SEQ ID NO: 29 in 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1413 EGFR VH 1414 EGFR VH 1415 EGFR VH 1416 EGFR VH 1417 EGFR VH 1418 EGFR VH 1419 EGFR VH 1420 EGFR VH 1421 EGFR VH 1422 EGFR VH 1423 EGFR VH 1424 EGFR VH 1425 EGFR VH 1426 EGFR VH 1427 EGFR VH 1428 EGFR VH 1429 EGFR VH 1430 EGFR VH 1431 EGFR VH 1432 EGFR VH 1433 EGFR VH 1434 EGFR VH 1435 EGFR VH 1436 EGFR VH 1437 EGFR VH 1438 EGFR VH 1439 EGFR VH 1440 EGFR VH 1441 EGFR VH 1442 EGFR VH 1443 EGFR VH 1444 EGFR VH 1445 EGFR VH 1446 EGFR VH 1447 EGFR VH 1448 EGFR VH 1449 EGFR VH 1450 EGFR VH 1451 EGFR VH 1452 EGFR VH 1453 EGFR VH 1454 EGFR VH 1455 EGFR VH 1456 EGFR VH 1457 EGFR VH 1458 EGFR VH 1459 EGFR VH 1460 EGFR VH 1461 EGFR VH 1462 EGFR VH 1463 EGFR VH 1464 EGFR VH 1465 EGFR VH 1466 EGFR VH 1467 EGFR VH 1468 EGFR VH 1469 EGFR VH 1470 EGFR VH 1471 EGFR VH 1472 EGFR VH 1473 EGFR VH 1474 EGFR VH 1475 EGFR VH 1476 EGFR VH 1477 EGFR VH 1478 EGFR VH 1479 EGFR VH 1480 EGFR VH 1481 EGFR VH 1482 EGFR VH 1483 EGFR VH 1484 EGFR VH 1485 EGFR VH 1486 EGFR VH 1487 EGFR VH 1488 EGFR VH 1489 EGFR VH 1490 EGFR VH 1491 EGFR VH 1492 EGFR VH 1493 EGFR VH 1494 EGFR VH 1495 EGFR VH 1496 EGFR VH 1497 EGFR VH 1498 EGFR VH 1499 EGFR VH 1500 EGFR VH 1501 EGFR VH 1502 EGFR VH 1503 EGFR VH 1504 EGFR VH 1505 EGFR VH 1506 EGFR VH 1507 EGFR VH 1508 EGFR VH 1509 EGFR VH 1510 EGFR VH 1511 EGFR VH 1512 EGFR VH 1513 EGFR VH 1514 EGFR VH 1515 EGFR VH 1516 EGFR VH 1517 EGFR VH 1518 EGFR VH 1519 EGFR VH 1520 EGFR VH 1521 EGFR VH 1522 EGFR VH 1523 EGFR VH 1524 EGFR VH 1525 EGFR VH 1526 EGFR VH 1527 EGFR VH 1528 EGFR VH 1529 EGFR VH 1530 EGFR VH 1531 EGFR VH 1532 EGFR VH 1533 EGFR VH 1534 EGFR VH 1535 EGFR VH 1536 EGFR VH 1537 EGFR VH 1538 EGFR VH 1539 EGFR VH 1540 EGFR VH 1541 EGFR VH 1542 EGFR VH 1543 EGFR VH 1544 EGFR VH 1545 EGFR VH 1546 EGFR VH 1547 EGFR VH 1548 EGFR VH 1549 EGFR VH 1550 EGFR VH 1551 EGFR VH 1552 EGFR VH 1553 EGFR VH 1554 EGFR VH 1555 EGFR VH 1556 EGFR VH 1557 EGFR VH 1558 EGFR VH 1559 EGFR VH 1560 EGFR VH 1561 EGFR VH 1562 EGFR VH 1563 EGFR VH 1564 EGFR VH 1565 EGFR VH 1566 EGFR VH 1567 EGFR VH 1568 EGFR VH 1569 EGFR VH 1570 EGFR VH 1571 EGFR VH 1572 EGFR VH 1573 EGFR VH 1574 EGFR VH 1575 EGFR VH 1576 EGFR VH 1577 EGFR VH 1578 EGFR VH 1579 EGFR VH 1580 EGFR VH 1581 EGFR VH 1582 EGFR VH 1583 EGFR VH 1584 EGFR VH 1585 EGFR VH 1586 EGFR VH 1587 EGFR VH 1588 EGFR VH 1589 EGFR VH 1590 EGFR VH 1591 EGFR VH 1592 EGFR VH 1593 EGFR VH 1594 EGFR VH 1595 EGFR VH 1596 EGFR VH 1597 EGFR VH 1598 EGFR VH 1599 EGFR VH 1600 EGFR VH 1601 EGFR VH 1602 EGFR VH 1603 EGFR VH 1604 EGFR VH 1605 EGFR VH 1606 EGFR VH 1607 EGFR VH 1608 EGFR VH 1609 EGFR VH 1610 EGFR VH 1611 EGFR VH 1612 EGFR VH 1613 EGFR VH 1614 EGFR VH 1615 EGFR VH 1616 EGFR VH 1617 EGFR VH 1618 EGFR VH 1619 EGFR VH 1620 EGFR VH 1621 EGFR VH 1622 EGFR VH 1623 EGFR VH 1624 EGFR VH 1625 EGFR VH 1626 EGFR VH 1627 EGFR VH 1628 EGFR VH 1629 EGFR VH 1630 EGFR VH 1631 EGFR VH 1632 EGFR VH 1633 EGFR VH 1634 EGFR VH 1635 EGFR VH 1636 EGFR VH 1637 EGFR VH 1638 EGFR VH 1639 EGFR VH 1640 EGFR VH 1641 EGFR VH 1642 EGFR VH 1643 EGFR VH 1644 EGFR VH 1645 EGFR VH 1646 EGFR VH 1647 EGFR VH 1648 EGFR VH 1649 EGFR VH 1650 EGFR VH 1651 EGFR VH 1652 EGFR VH 1653 EGFR VH 1654 EGFR VH 1655 EGFR VH 1656 EGFR VH 1657 EGFR VH 1658 EGFR VH 1659 EGFR VH 1660 EGFR VH 1661 EGFR VH 1662 EGFR VH 1663 EGFR VH 1664 EGFR VH 1665 EGFR VH 1666 EGFR VH 1667 EGFR VH 1668 EGFR VH 1669 EGFR VH 1670 EGFR VH 1671 EGFR VH 1672 EGFR VH 1673 EGFR VH 1674 EGFR VH 1675 EGFR VH 1676 EGFR VH 1677 EGFR VH 1678 EGFR VH 1679 EGFR VH 1680 EGFR VH 1681 EGFR VH 1682 EGFR VH 1683 EGFR VH 1684 EGFR VH 1685 EGFR VH 1686 EGFR VH 1687 EGFR VH 1688 EGFR VH 1689 EGFR VH 1690 EGFR VH 1691 EGFR VH 1692 EGFR VH 1693 EGFR VH 1694 EGFR VH 1695 EGFR VH 1696 EGFR VH 1697 EGFR VH 1698 EGFR VH 1699 EGFR VH 1700 EGFR VH 1701 EGFR VH 1702 EGFR VH 1703 EGFR VH 1704 EGFR VH 1705 EGFR VH 1706 EGFR VH 1707 EGFR VH 1708 EGFR VH 1709 EGFR VH 1710 EGFR VH 1711 EGFR VH 1712 EGFR VH 1713 EGFR VH 1714 EGFR VH 1715 EGFR VH 1716 EGFR VH 1717 EGFR VH 1718 EGFR VH 1719 EGFR VH 1720 EGFR VH 1721 EGFR VH 1722 EGFR VH 1723 EGFR VH 1724 EGFR VH 1725 EGFR VH 1726 EGFR VH 1727 EGFR VH 1728 EGFR VH 1729 EGFR VH 1730 EGFR VH 1731 EGFR VH 1732 EGFR VH 1733 EGFR VH 1734 EGFR VH 1735 EGFR VH 1736 EGFR VH 1737 EGFR VH 1738 EGFR VH 1739 EGFR VH 1740 EGFR VH 1741 EGFR VH 1742 EGFR VH 1743 EGFR VH 1744 EGFR VH 1745 EGFR VH 1746 EGFR VH 1747 EGFR VH 1748 EGFR VH 1749 EGFR VH 1750 EGFR VH 1751 EGFR VH 1752 EGFR VH 1753 EGFR VH 1754 EGFR VH 1755 EGFR VH 1756 EGFR VH 1757 EGFR VH 1758 EGFR VH 1759 EGFR VH 1760 EGFR VH 1761 EGFR VH 1762 EGFR VH 1763 EGFR VH 1764 EGFR VH 1765 EGFR VH 1766 EGFR VH 1767 EGFR VH 1768 EGFR VH 1769 EGFR VH 1770 EGFR VH 1771 EGFR VH 1772 EGFR VH 1773 EGFR VH 1774 EGFR VH 1775 EGFR VH 1776 EGFR VH 1777 EGFR VH 1778 EGFR VH 1779 EGFR VH 1780 EGFR VH 1781 EGFR VH 1782 EGFR VH 1783 EGFR VH 1784 EGFR VH 1785 EGFR VH 1786 EGFR VH 1787 EGFR VH 1788 EGFR VH 1789 EGFR VH 1790 EGFR VH 1791 EGFR VH 1792 EGFR VH 1793 EGFR VH 1794 EGFR VH 1795 EGFR VH 1796 EGFR VH 1797 EGFR VH 1798 EGFR VH 1799 EGFR VH 1800 EGFR VH 1801 EGFR VH 1802 EGFR VH 1803 EGFR VH 1804 EGFR VH 1805 EGFR VH 1806 EGFR VH 1807 EGFR VH 1808 EGFR VH 1809 EGFR VH 1810 EGFR VH 1811 EGFR VH 1812 EGFR VH 1813 EGFR VH 1814 EGFR VH 1815 EGFR VH 1816 EGFR VH 1817 EGFR VH 1818 EGFR VH 1819 EGFR VH 1820 EGFR VH 1821 EGFR VH 1822 EGFR VH 1823 EGFR VH 1824 EGFR VH 1825 EGFR VH 1826 EGFR VH 1827 EGFR VH 1828 EGFR VH 1829 EGFR VH 1830 EGFR VH 1831 EGFR VH 1832 EGFR VH 1833 EGFR VH 1834 EGFR VH 1835 EGFR VH 1836 EGFR VH 1837 EGFR VH 1838 EGFR VH 1839 EGFR VH 1840 EGFR VH 1841 EGFR VH 1842 EGFR VH 1843 EGFR VH 1844 EGFR VH 1845 EGFR VH 1846 EGFR VH 1847 EGFR VH 1848 EGFR VH 1849 EGFR VH 1850 EGFR VH 1851 EGFR VH 1852 EGFR VH 1853 EGFR VH 1854 EGFR VH 1855 EGFR VH 1856 EGFR VH 1857 EGFR VH 1858 EGFR VH 1859 EGFR VH 1860 EGFR VH 1861 EGFR VH 1862 EGFR VH 1863 EGFR VH 1864 EGFR VH 1865 EGFR VH 1866 EGFR VH 1867 EGFR VH 1868 EGFR VH 1869 EGFR VH 1870 EGFR VH 1871 EGFR VH 1872 EGFR VH 1873 EGFR VH 1874 EGFR VH 1875 EGFR VH 1876 EGFR VH 1877 EGFR VH 1878 EGFR VH 1879 EGFR VH 1880 EGFR VH 1881 EGFR VH 1882 EGFR VH 1883 EGFR VH 1884 EGFR VH 1885 EGFR VH 1886 EGFR VH 1887 EGFR VH 1888 EGFR VH 1889 EGFR VH 1890 EGFR VH 1891 EGFR VH 1892 EGFR VH 1893 EGFR VH 1894 EGFR VH 1895 EGFR VH 1896 EGFR VH 1897 EGFR VH 1898 EGFR VH 1899 EGFR VH 1900 EGFR VH 1901 EGFR VH 1902 EGFR VH 1903 EGFR VH 1904 EGFR VH 1905 EGFR VH 1906 EGFR VH 1907 EGFR VH 1908 EGFR VH 1909 EGFR VH 1910 EGFR VH 1911 EGFR VH 1912 EGFR VH 1913 EGFR VH 1914 EGFR VH 1915 EGFR VH 1916 EGFR VH 1917 EGFR VH 1918 EGFR VH 1919 EGFR VH 1920 EGFR VH 1921 EGFR VH 1922 EGFR VH 1923 EGFR VH 1924 EGFR VH 1925 EGFR VH 1926 EGFR VH 1927 EGFR VH 1928 EGFR VH 1929 EGFR VH 1930 EGFR VH 1931 EGFR VH 1932 EGFR VH 1933 EGFR VH 1934 EGFR VH 1935 EGFR VH 1936 EGFR VH 1937 EGFR VH 1938 EGFR VH 1939 EGFR VH 1940 EGFR VH 1941 EGFR VH 1942 EGFR VH 1943 EGFR VH 1944 EGFR VH 1945 EGFR VH 1946 EGFR VH 1947 EGFR VH 1948 EGFR VH 1949 EGFR VH 1950 EGFR VH 1951 EGFR VH 1952 EGFR VH 1953 EGFR VH 1954 EGFR VH 1955 EGFR VH 1956 EGFR VH 1957 EGFR VH 1958 EGFR VH 1959 EGFR VH 1960 EGFR VH 1961 EGFR VH 1962 EGFR VH 1963 EGFR VH 1964 EGFR VH 1965 EGFR VH 1966 EGFR VH 1967 EGFR VH 1968 EGFR VH 1969 EGFR VH 1970 EGFR VH 1971 EGFR VH 1972 EGFR VH 1973 EGFR VH 1974 EGFR VH 1975 EGFR VH 1976 EGFR VH 1977 EGFR VH 1978 EGFR VH 1979 EGFR VH 1980 EGFR VH 1981 EGFR VH 1982 EGFR VH 1983 EGFR VH 1984 EGFR VH 1985 EGFR VH 1986 EGFR VH 1987 EGFR VH 1988 EGFR VH 1989 EGFR VH 1990 EGFR VH 1991 EGFR VH 1992 EGFR VH 1993 EGFR VH 1994 EGFR VH 1995 EGFR VH 1996 EGFR VH 1997 EGFR VH 1998 EGFR VH 1999 EGFR VH 2000 EGFR VH 2001 EGFR VH 2002 EGFR VH 2003 EGFR VH 2004 EGFR VH 2005 EGFR VH 2006 EGFR VH 2007 EGFR VH 2008 EGFR VH 2009 EGFR VH 2010 EGFR VH 2011 EGFR VH 2012 EGFR VH 2013 EGFR VH 2014 EGFR VH 2015 EGFR VH 2016 EGFR VH 2017 EGFR VH 2018 EGFR VH 2019 EGFR VH 2020 EGFR VH 2021 EGFR VH 2022 EGFR VH 2023 EGFR VH 2024 EGFR VH 2025 EGFR VH 2026 EGFR VH 2027 EGFR VH 2028 EGFR VH 2029 EGFR VH 2030 EGFR VH 2031 EGFR VH 2032 EGFR VH 2033 EGFR VH 2034 EGFR VH 2035 EGFR VH 2036 EGFR VH 2037 EGFR VH 2038 EGFR VH 2039 EGFR VH 2040 EGFR VH 2041 EGFR VH 2042 EGFR VH 2043 EGFR VH 2044 EGFR VH 2045 EGFR VH 2046 EGFR VH 2047 EGFR VH 2048 EGFR VH 2049 EGFR VH 2050 EGFR VH 2051 EGFR VH 2052 EGFR VH 2053 EGFR VH 2054 EGFR VH 2055 EGFR VH 2056 EGFR VH 2057 EGFR VH 2058 EGFR VH 2059 EGFR VH 2060 EGFR VH 2061 EGFR VH 2062 EGFR VH 2063 EGFR VH 2064 EGFR VH 2065 EGFR VH 2066 EGFR VH 2067 EGFR VH 2068 EGFR VH 2069 EGFR VH 2070 EGFR VH 2071 EGFR VH 2072 EGFR VH 2073 EGFR VH 2074 EGFR VH 2075 EGFR VH 2076 EGFR VH 2077 EGFR VH 2078 EGFR VH 2079 EGFR VH 2080 EGFR VH 2081 EGFR VH 2082 EGFR VH 2083 EGFR VH 2084 EGFR VH 2085 EGFR VH 2086 EGFR VH 2087 EGFR VH 2088 EGFR VH 2089 EGFR VH 2090 EGFR VH 2091 EGFR VH 2092 EGFR VH 2093 EGFR VH 2094 EGFR VH 2095 EGFR VH 2096 EGFR VH 2097 EGFR VH 2098 EGFR VH 2099 EGFR VH 2100 EGFR VH 2101 EGFR VH 2102 EGFR VH 2103 EGFR VH 2104 EGFR VH 2105 EGFR VH 2106 EGFR VH 2107 EGFR VH 2108 EGFR VH 2109 EGFR VH 2110 EGFR VH 2111 EGFR VH 2112 EGFR VH 2113 EGFR VH 2114 EGFR VH 2115 EGFR VH 2116 EGFR VH 2117 EGFR VH 2118 EGFR VH 2119 EGFR VH 2120 EGFR VH 2121 EGFR VH 2122 EGFR(Cetuximab) VH 2123 EGFR(Cetuximab) VH 2124 EGFR(EGFRvIII) VH 2125 EGFR(EGFRvIII) VH 2126 EGFR(EGFRvIII) VH 2127 EGFR(EGFRvIII) VH 2128 EGFR(EGFRvIII) VH 2129 EGFR(EGFRvIII) VH 2130 EGFR(EGFRvIII) VH 2131 EGFR(EGFRvIII) VH 2132 EGFR(EGFRvIII) VH 2133 EGFR(EGFRvIII) VH 2134 EGFR(EGFRvIII) VH 2135 EGFR(EGFRvIII) VH 2136 EGFR(EGFRvIII) VH 2137 EGFR(EGFRvIII) VH 2138 EGFR(EGFRvIII) VH 2139 EGFR(EGFRvIII) VH 2140 EGFR(EGFRvIII) VH 2141 EGFR(EGFRvIII) VH 2142 EGFR(EGFRvIII) VH 2143 EGFR(EGFRvIII) VH 2144 EGFR(EGFRvIII) VH 2145 EGFR(EGFRvIII) VH 2146 EGFRvIII VH 2147 SEQ ID NO. 13 in WO2016016341 EGFRvIII VH 2148 SEQ ID NO: 24 in WO2016168773A3 EGFRvIII VH 2149 SEQ ID NO. 34 in US20160304615 EGFRvIII VH 2150 SEQ ID NO: 2 in US20160200819A1 Endoglin VH 2151 SEQ ID NO. 41 in US20160009811 Endoglin VH 2152 SEQ ID NO. 42 in US20160009811 Endoglin VH 2153 SEQ ID NO. 43 in US20160009811 Endoglin VH 2154 SEQ ID NO. 71 in US20160009811 Endoglin VH 2155 SEQ ID NO. 73 in US20160009811 Endoglin VH 2156 SEQ ID NO. 75 in US20160009811 Endoglin VH 2157 SEQ ID NO. 88 in US20160009811 Endoglin VH 2158 SEQ ID NO. 89 in US20160009811 Endoglin VH 2159 SEQ ID NO. 90 in US20160009811 Endoglin VH 2160 SEQ ID NO. 91 in US20160009811 Endoglin VH 2161 SEQ ID NO. 92 in US20160009811 EphA2receptor VH 2162 US20150274824 SEQ ID NO: 20 EphA2receptor VH 2163 US20150274824 SEQ ID NO: 22 EphA2receptor VH 2164 US20150274824 SEQ ID NO: 24 EphA2receptor VH 2165 US20150274824 SEQ ID NO: 32 EphA2receptor VH 2166 US20150274824 SEQ ID NO: 34 EphA2receptor VH 2167 US20150274824 SEQ ID NO: 36 EphA2receptor VH 2168 US20150274824 SEQ ID NO: 37 EphA2receptor VH 2169 US20150274824 SEQ ID NO: 38 EphA2receptor VH 2170 US20150274824 SEQ ID NO: 40 EphA2receptor VH 2171 US20150274824 SEQ ID NO: 42 EphA2receptor VH 2172 US20150274824 SEQ ID NO: 43 EphA2receptor VH 2173 US20150274824 SEQ ID NO: 45 EphA2receptor VH 2174 US20150274824 SEQ ID NO: 74 EphA2receptor VH 2175 US20150274824 SEQ ID NO: 76 ERBB2 VH 2176 US20110129464 SEQ ID NO: 2 ERBB2 VH 2177 US20110129464 SEQ ID NO: 4 ERBB2 VH 2178 US20130089544 SEQ ID NO: 10 ERBB2 VH 2179 US20130089544 SEQ ID NO: 2 ERBB2 VH 2180 US20130089544 SEQ ID NO: 26 ERBB2 VH 2181 US20130089544 SEQ ID NO: 30 ERBB2 VH 2182 US20130089544 SEQ ID NO: 38 ERBB2 VH 2183 US20130089544 SEQ ID NO: 4 ERBB2 VH 2184 US20130089544 SEQ ID NO: 40 ERBB2 VH 2185 US20130089544 SEQ ID NO: 42 ERBB2 VH 2186 US20130089544 SEQ ID NO: 52 ERBB2 VH 2187 US20130089544 SEQ ID NO: 54 ERBB2 VH 2188 US20130089544 SEQ ID NO: 56 ERBB2 VH 2189 US20130089544 SEQ ID NO: 57 ERBB2 VH 2190 US20130089544 SEQ ID NO: 58 ERBB2 VH 2191 US20130089544 SEQ ID NO: 6 ERBB2 VH 2192 US20130266564 SEQ ID NO: 8 ERBB2 VH 2193 US20150104443 SEQ ID NO: 1 FactorD VH 2194 SEQ ID NO. 17 in US20160017052 FactorD VH 2195 SEQ ID NO. 20 in US20160017052 FactorD VH 2196 SEQ ID NO. 27 in US20160017052 FactorD VH 2197 SEQ ID NO. 29 in US20160017052 FactorD VH 2198 SEQ ID NO. 30 in US20160017052 FactorD VH 2199 SEQ ID NO. 31 in US20160017052 FactorD VH 2200 SEQ ID NO. 32 in US20160017052 FactorD VH 2201 SEQ ID NO. 33 in US20160017052 FactorD VH 2202 SEQ ID NO. 4 in US20160017052 FactorXII VH 2203 SEQ ID NO. 15 in WO2014089493 FAP VH 2204 SEQ ID NO. 1 in WO2015118030 FAP VH 2205 SEQ ID NO. 5 in WO2015118030 FAP VH 2206 SEQ ID NO. 170 in WO2016120216 FAP VH 2207 SEQ ID NO. 172 in WO2016120216 FcRL5(FcReceptorLike5) VH 2208 SEQ ID NO: 12 WO2016090337 FcRL5(FcReceptorLike5) VH 2209 SEQ ID NO: 16 WO2016090337 FcRL5(FcReceptorLike5) VH 2210 SEQ ID NO: 20 WO2016090337 FcRL5(FcReceptorLike5) VH 2211 SEQ ID NO: 24 WO2016090337 FcRL5(FcReceptorLike5) VH 2212 SEQ ID NO: 28 WO2016090337 FcRL5(FcReceptorLike5) VH 2213 SEQ ID NO: 32 WO2016090337 FcRL5(FcReceptorLike5) VH 2214 SEQ ID NO: 36 WO2016090337 FcRL5(FcReceptorLike5) VH 2215 SEQ ID NO: 4 WO2016090337 FcRL5(FcReceptorLike5) VH 2216 SEQ ID NO: 40 WO2016090337 FcRL5(FcReceptorLike5) VH 2217 SEQ ID NO: 44 WO2016090337 FcRL5(FcReceptorLike5) VH 2218 SEQ ID NO: 48 WO2016090337 FcRL5(FcReceptorLike5) VH 2219 SEQ ID NO: 8 WO2016090337 FcRL5(FcReceptorLike5) VH 2220 SEQ ID NO: 915 WO2016090337 FcRL5(FcReceptorLike5) VH 2221 SEQ ID NO: 919 WO2016090337 FGFR3 VH 2222 SEQ ID NO. 132 in U.S. Pat. No. 9,499,623 FGFR3 VH 2223 SEQ ID NO. 134 in U.S. Pat. No. 9,499,623 FGFR3 VH 2224 SEQ ID NO. 136 in U.S. Pat. No. 9,499,623 FGFR4 VH 2225 SEQ ID NO. 7 in US20160237157 Frizzled Receptor VH 2226 SEQ ID NO. 10 in WO2010037041 GAH VH 2227 SEQ ID NO 7 in US20060057147A1 GCC1 VH 2228 SEQ ID NO. 1 in US20160030595A1 GD2 VH 2229 SEQ ID NO. 10 in WO2015132604 GD2 VH 2230 SEQ ID NO. 3 in US20130216528 GD2 VH 2231 SEQ ID NO. 4 in US20130216528 GD2 VH 2232 SEQ ID NO. 6 in US20130216528 GD2 VH 2233 SEQ ID NO. 8 in US20130216528 GD2 VH 2234 SEQ ID NO. 9 in WO2015132604 GD3 VH 2235 SEQ ID NO: 11 in WO2016185035A1 GD3 VH 2236 SEQ ID NO: 13 in WO2016185035A1 GD3 VH 2237 SEQ ID NO: 15 in WO2016185035A1 GD3 VH 2238 SEQ ID NO: 17 in WO2016185035A1 Glyco epitope and ErbBB I VH 2239 SEQ ID No. 7 in WO2012007167A1 Specific Glyco epitope and ErbBBI VH 2240 SEQ ID No. 9 in WO2012007167A1 Specific GM2 VH 2241 US20090028877 SEQ ID NO: 20 GM2 VH 2242 US20090028877 SEQ ID NO: 22 GM2 VH 2243 US20090028877 SEQ ID NO: 23 GM2 VH 2244 US20090028877 SEQ ID NO: 26 GM2 VH 2245 US20090028877 SEQ ID NO: 27 GM2 VH 2246 US20090028877 SEQ ID NO: 28 GM2 VH 2247 US20090028877 SEQ ID NO: 29 GM2 VH 2248 US20090028877 SEQ ID NO: 30 GPC3 VH 2249 SEQ ID NO: 10 in US20160208015A1 GPC3 VH 2250 SEQ ID NO: 14 in US20160208015A1 GPC3 VH 2251 SEQ ID NO: 2 in US20160208015A1 GPC3 VH 2252 SEQ ID NO: 3 in US20160208015A1 GPC3 VH 2253 SEQ ID NO: 4 in US20160208015A1 GPC3 VH 2254 SEQ ID NO: 5 in US20160208015A1 GPC3 VH 2255 SEQ ID NO: 6 in US20160208015A1 GPC3 VH 2256 SEQ ID NO: 7 in US20160208015A1 GPC3 VH 2257 SEQ ID NO: 8 in US20160208015A1 GPC3 VH 2258 SEQ ID NO: 9 in US20160208015A1 GPRC5D VH 2259 SEQ ID NO. 13 in WO2016090312 GPRC5D VH 2260 SEQ ID NO. 17 in WO2016090312 GPRC5D VH 2261 SEQ ID NO. 21 in WO2016090312 GPRC5D VH 2262 SEQ ID NO. 25 in WO2016090312 GPRC5D VH 2263 SEQ ID NO. 29 in WO2016090312 GPRC5D VH 2264 SEQ ID NO. 314 in WO2016090312 GPRC5D VH 2265 SEQ ID NO. 326 in WO2016090312 GPRC5D VH 2266 SEQ ID NO. 33 in WO2016090312 GPRC5D VH 2267 SEQ ID NO. 338 in WO2016090312 GPRC5D VH 2268 SEQ ID NO. 350 in WO2016090312 GPRC5D VH 2269 SEQ ID NO. 362 in WO2016090312 GPRC5D VH 2270 SEQ ID NO. 37 in WO2016090312 GPRC5D VH 2271 SEQ ID NO. 374 in WO2016090312 GPRC5D VH 2272 SEQ ID NO. 386 in WO2016090312 GPRC5D VH 2273 SEQ ID NO. 41 in WO2016090312 GPRC5D VH 2274 SEQ ID NO. 45 in WO2016090312 GPRC5D VH 2275 SEQ ID NO. 49 in WO2016090312 GPRC5D VH 2276 SEQ ID NO. 5 in WO2016090312 GPRC5D VH 2277 SEQ ID NO. 53 in WO2016090312 GPRC5D VH 2278 SEQ ID NO. 57 in WO2016090312 GPRC5D VH 2279 SEQ ID NO. 61 in WO2016090312 GPRC5D VH 2280 SEQ ID NO. 65 in WO2016090312 GPRC5D VH 2281 SEQ ID NO. 69 in WO2016090312 GPRC5D VH 2282 SEQ ID NO. 73 in WO2016090312 GPRC5D VH 2283 SEQ ID NO. 77 in WO2016090312 GPRC5D VH 2284 SEQ ID NO. 81 in WO2016090312 GPRC5D VH 2285 SEQ ID NO. 85 in WO2016090312 GPRC5D VH 2286 SEQ ID NO. 89 in WO2016090312 GPRC5D VH 2287 SEQ ID NO. 93 in WO2016090312 GPRC5D VH 2288 SEQ ID NO. 1 in WO2016090312 GPRC5D VH 2289 SEQ ID NO. 9 in WO2016090312 Her1/her3 VH 2290 SEQ ID NO: 8 of WO2016073629 Her2 VH 2291 SEQ ID NO: 141 in WO2016054555A2 Her2 VH 2292 SEQ ID NO: 262 in WO2016168773A3 Her2 VH 2293 SEQ ID NO: 264 in WO2016168773A3 Her2 VH 2294 SEQ ID NO: 266 in WO2016168773A3 Her2 VH 2295 SEQ ID NO: 268 in WO2016168773A3 Her2 VH 2296 SEQ ID NO: 270 in WO2016168773A3 HER2 VH 2297 SEQ ID NO. 11 in U.S. Pat. No. 9,518,118 HER2 VH 2298 SEQ ID NO: 62 in US20160333114A1 HLAG VH 2299 SEQ ID NO. 10 in WO2016160622A2 HLAG VH 2300 SEQ ID NO. 8 in WO2016160622A2 HSP70 VH 2301 SEQ ID NO. 11 in WO2016120217 HSP70 VH 2302 SEQ ID NO. 12 in WO2016120217 humanCD79b VH 2303 SEQ ID NO. 27 in WO2016112870 humanCD79b VH 2304 SEQ ID NO. 29 in WO2016112870 Human chorionic VH 2305 SEQ ID NO. 2 in WO2007019541 gonadotropin Human chorionic VH 2306 SEQ ID NO. 4 in WO2007019541 gonadotropin Human chorionic VH 2307 SEQ ID NO. 6 in WO2007019541 gonadotropin Human collagen VII VH 2308 SEQ ID NO. 31 in WO2016112870 humanERBB3 VH 2309 SEQ ID NO: 19 in WO2013052745 humanERBB3 VH 2310 SEQ ID NO: 29 in WO2013052745 humanERBB3 VH 2311 SEQ ID NO: 38 in WO2013052745 humanERBB3 VH 2312 SEQ ID NO: 45 in WO2013052745 humanERBB3 VH 2313 SEQ ID NO: 55 in WO2013052745 humanERBB3 VH 2314 SEQ ID NO: 61 in WO2013052745 humanERBB3 VH 2315 SEQ ID NO: 9 in WO2013052745 ICOS VH 2316 SEQ ID NO. 15 in US20160215059 ICOS VH 2317 SEQ ID NO. 16 in US20160215059 ICOS VH 2318 SEQ ID NO. 19 in US20160215059 ICOS VH 2319 SEQ ID NO. 23 in US20160215059 ICOS VH 2320 SEQ ID NO. 7 in US20160215059 IGFI VH 2321 SEQ ID NO. 1 in WO2007118214 IGFI VH 2322 SEQ ID NO. 3 in WO2007118214 IGFI VH 2323 SEQ ID NO. 7 in WO2007118214 IGFR1 VH 2324 SEQ ID NO: 7 in WO2015073575A2 IL13 VH 2325 SEQ ID NO 302. in US20160168242 IL13Ra2 VH 2326 SEQ ID NO. 7 in WO2016123143 IL13Ra2 VH 2327 SEQ ID NO. 8 in WO2016123143 IL1RAP VH 2328 SEQ ID NO. 1 in WO2016020502 IL1RAP VH 2329 SEQ ID NO. 10 in WO2016020502 IL1RAP VH 2330 SEQ ID NO. 19 in WO2016020502 IL1RAP VH 2331 SEQ ID NO. 8 in WO2016020502 IL1RAP VH 2332 SEQ ID NO. 9 in WO2016020502 IL1RAP VH 2333 SEQ ID NO: 120 in WO2016179319A1 IL1RAP VH 2334 SEQ ID NO: 122 in WO2016179319A1 IL1RAP VH 2335 SEQ ID NO: 124 in WO2016179319A1 IL21 VH 2336 SEQ ID NO. 2 in US20160145332 IL21 VH 2337 SEQ ID NO. 3 in US20160145332 IL33 VH 2338 SEQ ID NO 134. in US20160168242 IL33 VH 2339 SEQ ID NO 136. in US20160168242 IL33 VH 2340 SEQ ID NO 138. in US20160168242 IL33 VH 2341 SEQ ID NO 183. in US20160168242 IL33 VH 2342 SEQ ID NO 185. in US20160168242 IL33 VH 2343 SEQ ID NO 187. in US20160168242 IL33 VH 2344 SEQ ID NO 189. in US20160168242 IL33 VH 2345 SEQ ID NO 216. in US20160168242 IL33 VH 2346 SEQ ID NO 218. in US20160168242 IL33 VH 2347 SEQ ID NO 220. in US20160168242 IL33 VH 2348 SEQ ID NO 221. in US20160168242 IL33 VH 2349 SEQ ID NO 236. in US20160168242 IL33 VH 2350 SEQ ID NO 246. in US20160168242 IL33 VH 2351 SEQ ID NO 282. in US20160168242 IL33 VH 2352 SEQ ID NO 284. in US20160168242 IL33 VH 2353 SEQ ID NO 286. in US20160168242 IL33 VH 2354 SEQ ID NO 36. in US20160168242 IL33 VH 2355 SEQ ID NO 38. in US20160168242 IL33 VH 2356 SEQ ID NO 40. in US20160168242 IL33 VH 2357 SEQ ID NO 84. in US20160168242 IL33 VH 2358 SEQ ID NO 86. in US20160168242 IL33 VH 2359 SEQ ID NO 88. in US20160168242 IL3alpha VH 2360 SEQ ID NO. 22 in WO2008127735 Integrin VH 2361 SEQ ID NO. 3 in US 20140161794 Integrin VH 2362 SEQ ID NO. 4 in US 20140161794 Integrin VH 2363 SEQ ID NO. 5 in US 20140161794 KDR VH 2364 SEQ ID NO. 20 IN WO2003075840 KDR VH 2365 SEQ ID NO. 24 IN WO2003075840 KDR VH 2366 SEQ ID NO. 26 IN WO2003075840 KDR VH 2367 SEQ ID NO. 29 IN WO2003075840 KDR VH 2368 SEQ ID NO. 31 IN WO2003075840 KDR VH 2369 SEQ ID NO. 33 IN WO2003075840 KIR(Lirilumab) VH 2370 SEQ ID NO. 3 in US20150290316 KIR(Lirilumab) VH 2371 SEQ ID NO. 1 in WO2014055648 KIR2DL1andKIR2DL2/3 VH 2372 SEQIDNO: 36 in WO2016126213A1 Klon43 VH 2373 SEQ ID NO: 47 in WO2016097231 KMA VH 2374 SEQ ID NO: 22 in WO2016172703A2 LAG3 VH 2375 SEQ ID NO. 100 in US20150259420 LAG3 VH 2376 SEQ ID NO. 104 in US20150259420 LAG3 VH 2377 SEQ ID NO. 108 in US20150259420 LAG3 VH 2378 SEQ ID NO. 28 in US20150259420 LAG3 VH 2379 SEQ ID NO. 64 in US20150259420 LAG3 VH 2380 SEQ ID NO. 68 in US20150259420 LAG3 VH 2381 SEQ ID NO. 72 in US20150259420 LAG3 VH 2382 SEQ ID NO. 76 in US20150259420 LAG3 VH 2383 SEQ ID NO. 8 in US20150259420 LAG3 VH 2384 SEQ ID NO. 80 in US20150259420 LAG3 VH 2385 SEQ ID NO. 1 in WO2015042246 leukocytegenA0 VH 2386 SEQ ID NO. 9 in WO2010065962A2 leukocytegenA2 VH 2387 SEQ ID NO. 25 in WO2010065962A2 LGR4 VH 2388 SEQ ID NO. 12 in US20160046723 LGR4 VH 2389 SEQ ID NO. 13 in US20160046723 LGR4 VH 2390 SEQ ID NO. 5 in US20160046723 LGR4 VH 2391 SEQ ID NO. 9 in US20160046723 LGR5 VH 2392 SEQ ID NO. 10 in US20160102146 LGR5 VH 2393 SEQ ID NO. 12 in US20160102146 LGR5 VH 2394 SEQ ID NO. 16 in US20160102146 LGR5 VH 2395 SEQ ID NO. 18 in US20160102146 LGR5 VH 2396 SEQ ID NO. 20 in US20160102146 LGR5 VH 2397 SEQ ID NO. 22 in US20160102146 LGR5 VH 2398 SEQ ID NO. 24 in US20160102146 LGR5 VH 2399 SEQ ID NO. 26 in US20160102146 LGR5 VH 2400 SEQ ID NO. 4 in US20160102146 LHR VH 2401 SEQ ID NO: 1 in WO2016160618A3 LHR VH 2402 SEQ ID NO: 2 in WO2016160618A3 LHR VH 2403 SEQ ID NO: 3 in WO2016160618A3 LHR VH 2404 SEQ ID NO: 4 in WO2016160618A3 LHR VH 2405 SEQ ID NO: 5 in WO2016160618A3 LHR VH 2406 SEQ ID NO: 6 in WO2016160618A3 LHR VH 2407 SEQ ID NO: 7 in WO2016160618A3 LHR VH 2408 SEQ ID NO: 8 in WO2016160618A3 lL4R VH 2409 SEQ ID NO. 10 in WO2009121847 lL4R VH 2410 SEQ ID NO. 11 in WO2009121847 lL4R VH 2411 SEQ ID NO. 14 in WO2009121847 lL4R VH 2412 SEQ ID NO. 15 in WO2009121847 lL4R VH 2413 SEQ ID NO. 9 in WO2009121847 Lymphotoxin beta receptor VH 2414 SEQ ID NO. 10 in WO2004002431 Lymphotoxin beta receptor VH 2415 SEQ ID NO. 12 in WO2004002431 Lymphotoxin beta receptor VH 2416 SEQ ID NO. 14 in WO2004002431 Lymphotoxin beta receptor VH 2417 SEQ ID NO. 16 in WO2004002431 Lymphotoxin beta receptor VH 2418 SEQ ID NO. 2 in WO2004002431 Lysyloxidaselike2 VH 2419 SEQ ID NO. 42 in WO2011097513 Lysyloxidaselike2 VH 2420 SEQ ID NO. 44 in WO2011097513 Malignant Variable Receptor VH 2421 SEQ ID NO. 1 in WO2015133817A1 MCAM VH 2422 SEQ ID NO. 115 in US20150259419 MCAM VH 2423 SEQ ID NO. 116 in US20150259419 MCAM VH 2424 SEQ ID NO. 117 in US20150259419 MCAM VH 2425 SEQ ID NO. 118 in US20150259419 MCAM VH 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US20170000901 CLL3 VL 3881 SEQ ID NO. 156 in US20170000901 CLL3 VL 3882 SEQ ID NO. 158 in US20170000901 CLL3 VL 3883 SEQ ID NO. 160 in US20170000901 CLL3 VL 3884 SEQ ID NO. 162 in US20170000901 CLL3 VL 3885 SEQ ID NO. 164 in US20170000901 CLL3 VL 3886 SEQ ID NO. 166 in US20170000901 CLL3 VL 3887 SEQ ID NO. 170 in US20170000901 CLL3 VL 3888 SEQ ID NO. 172 in US20170000901 CLL3 VL 3889 SEQ ID NO. 174 in US20170000901 CLL3 VL 3890 SEQ ID NO. 176 in US20170000901 CLL3 VL 3891 SEQ ID NO. 178 in US20170000901 CLL3 VL 3892 SEQ ID NO. 180 in US20170000901 CLL3 VL 3893 SEQ ID NO. 182 in US20170000901 CLL3 VL 3894 SEQ ID NO. 184 in US20170000901 CLL3 VL 3895 SEQ ID NO. 186 in US20170000901 CLL3 VL 3896 SEQ ID NO. 190 in US20170000901 CLL3 VL 3897 SEQ ID NO. 192 in US20170000901 CLL3 VL 3898 SEQ ID NO. 194 in US20170000901 CLL3 VL 3899 SEQ ID NO. 196 in US20170000901 CLL3 VL 3900 SEQ ID NO. 198 in US20170000901 CLL3 VL 3901 SEQ ID NO. 20 in US20170000901 CLL3 VL 3902 SEQ ID NO. 200 in US20170000901 CLL3 VL 3903 SEQ ID NO. 202 in US20170000901 CLL3 VL 3904 SEQ ID NO. 204 in US20170000901 CLL3 VL 3905 SEQ ID NO. 206 in US20170000901 CLL3 VL 3906 SEQ ID NO. 208 in US20170000901 CLL3 VL 3907 SEQ ID NO. 210 in US20170000901 CLL3 VL 3908 SEQ ID NO. 212 in US20170000901 CLL3 VL 3909 SEQ ID NO. 22 in US20170000901 CLL3 VL 3910 SEQ ID NO. 24 in US20170000901 CLL3 VL 3911 SEQ ID NO. 26 in US20170000901 CLL3 VL 3912 SEQ ID NO. 28 in US20170000901 CLL3 VL 3913 SEQ ID NO. 30 in US20170000901 CLL3 VL 3914 SEQ ID NO. 32 in US20170000901 CLL3 VL 3915 SEQ ID NO. 34 in US20170000901 CLL3 VL 3916 SEQ ID NO. 36 in US20170000901 CLL3 VL 3917 SEQ ID NO. 38 in US20170000901 CLL3 VL 3918 SEQ ID NO. 40 in US20170000901 CLL3 VL 3919 SEQ ID NO. 42 in US20170000901 CLL3 VL 3920 SEQ ID NO. 44 in US20170000901 CLL3 VL 3921 SEQ ID NO. 46 in US20170000901 CLL3 VL 3922 SEQ ID NO. 48 in US20170000901 CLL3 VL 3923 SEQ ID NO. 50 in US20170000901 CLL3 VL 3924 SEQ ID NO. 54 in US20170000901 CLL3 VL 3925 SEQ ID NO. 56 in US20170000901 CLL3 VL 3926 SEQ ID NO. 58 in US20170000901 CLL3 VL 3927 SEQ ID NO. 60 in US20170000901 CLL3 VL 3928 SEQ ID NO. 62 in US20170000901 CLL3 VL 3929 SEQ ID NO. 64 in US20170000901 CLL3 VL 3930 SEQ ID NO. 66 in US20170000901 CLL3 VL 3931 SEQ ID NO. 68 in US20170000901 CLL3 VL 3932 SEQ ID NO. 70 in US20170000901 CLL3 VL 3933 SEQ ID NO. 72 in US20170000901 CLL3 VL 3934 SEQ ID NO. 74 in US20170000901 CLL3 VL 3935 SEQ ID NO. 76 in US20170000901 CLL3 VL 3936 SEQ ID NO. 78 in US20170000901 CLL3 VL 3937 SEQ ID NO. 80 in US20170000901 CLL3 VL 3938 SEQ ID NO. 82 in US20170000901 CLL3 VL 3939 SEQ ID NO. 84 in US20170000901 CLL3 VL 3940 SEQ ID NO. 86 in US20170000901 CLL3 VL 3941 SEQ ID NO. 88 in US20170000901 CLL3 VL 3942 SEQ ID NO. 90 in US20170000901 CLL3 VL 3943 SEQ ID NO. 92 in US20170000901 CLL3 VL 3944 SEQ ID NO. 94 in US20170000901 CLL3 VL 3945 SEQ ID NO. 96 in US20170000901 CLL3 VL 3946 SEQ ID NO. 98 in US20170000901 collagen VL 3947 SEQ ID NO. 11 in WO2007024921 collagen VL 3948 SEQ ID NO. 12 in WO2007024921 collagen VL 3949 SEQ ID NO. 14 in WO2007024921 collagen VL 3950 SEQ ID NO. 23 in WO2007024921 collagen VL 3951 SEQ ID NO. 25 in WO2007024921 collagen VL 3952 SEQ ID NO. 26 in WO2007024921 collagen VL 3953 SEQ ID NO. 27 in WO2007024921 collagen VL 3954 SEQ ID NO. 8 in WO2007024921 collagen VL 3955 SEQ ID NO. 9 in WO2007024921 CS1 VL 3956 SEQ ID NO. 104 in WO2016120216 CS1 VL 3957 SEQ ID NO. 106 in WO2016120216 CS1 VL 3958 SEQ ID NO. 108 in WO2016120216 CS1 VL 3959 SEQ ID NO. 14 in WO2015166056A1 CS1 VL 3960 SEQ ID NO. 16 in WO2015166056A1 CS1 VL 3961 SEQ ID NO. 18 in WO2015166056A1 CS1 VL 3962 SEQ ID NO. 20 in WO2015166056A1 CS1 VL 3963 SEQ ID NO. 22 in WO2015166056A1 CS1 VL 3964 SEQ ID No. 39 in WO2015121454 CS1 VL 3965 SEQ ID No. 41 in WO2015121454 CS1 VL 3966 SEQ ID No. 43 in WO2015121454 CS1 VL 3967 SEQ ID No. 45 in WO2015121454 CS1 VL 3968 SEQ ID No. 47 in WO2015121454 CSF VL 3969 SEQ ID NO 12 in US20050059113A1 CSF VL 3970 SEQ ID NO 32 in US20050059113A1 CSF VL 3971 SEQ ID NO 44 in US20050059113A1 CSF VL 3972 SEQ ID NO 48 in US20050059113A1 CSF VL 3973 SEQ ID NO 60 in US20050059113A1 CSPG4 VL 3974 SEQ ID NO. 7 in WO2016164429 CTLA4 VL 3975 SEQ ID NO. 36 in US20140105914 CTLA4 VL 3976 SEQ ID NO. 37 in US20140105914 CTLA4 VL 3977 SEQ ID NO. 38 in US20140105914 CTLA4 VL 3978 SEQ ID NO. 39 in US20140105914 CTLA4 VL 3979 SEQ ID NO. 40 in US20140105914 CTLA4 VL 3980 SEQ ID NO. 46 in US20140105914 CTLA4 VL 3981 SEQ ID NO. 47 in US20140105914 CTLA4 VL 3982 SEQ ID NO. 48 in US20140105914 CTLA4 VL 3983 SEQ ID NO. 49 in US20140105914 CTLA4 VL 3984 SEQ ID NO. 50 in US20140105914 CTLA4 VL 3985 SEQ ID NO. 8 in US20140105914 CTLA4 VL 3986 SEQ ID NO. 2 in U.S. Pat. No. 8,697,845 CTLA4 VL 3987 SEQ ID NO. 4 in US20140105914 CTLA4(Ipilimumab) VL 3988 SEQ ID NO. 20 in US20150283234 CTLA4(Ipilimumab) VL 3989 SEQ ID NO. 18 in WO2014066532 CXCR4 VL 3990 US20110020218 SEQ ID NO: 76 CXCR4 VL 3991 US20110020218 SEQ ID NO: 77 CXCR4 VL 3992 US20110020218 SEQ ID NO: 78 CXCR4 VL 3993 US20110020218 SEQ ID NO: 79 CXCR4 VL 3994 US20110020218 SEQ ID NO: 80 CXCR4 VL 3995 US20110020218 SEQ ID NO: 81 CXCR4 VL 3996 US20110020218 SEQ ID NO: 82 CXCR4 VL 3997 US20110020218 SEQ ID NO: 87 CXCR4 VL 3998 US20110020218 SEQ ID NO: 88 CXCR4 VL 3999 US20110020218 SEQ ID NO: 90 CXCR4 VL 4000 US20110020218 SEQ ID NO: 91 CXCR4 VL 4001 US20110020218 SEQ ID NO: 92 CXCR4 VL 4002 US20110020218 SEQ ID NO: 93 Daclizumab VL 4003 SEQ ID NO: 43 in US20160333114A1 Daclizumab VL 4004 SEQ ID NO: 45 in US20160333114A1 DR5 VL 4005 SEQ ID NO. 13 in WO2016122701 DR5 VL 4006 SEQ ID NO. 23 in WO2016122701 DR5 VL 4007 SEQ ID NO. 25 in WO2016122701 DR5 VL 4008 SEQ ID NO. 27 in WO2016122701 DR5 VL 4009 SEQ ID NO. 3 in WO2016122701 DR5 VL 4010 SEQ ID NO. 78 in WO2016122701 DR5 VL 4011 SEQ ID NO. 86 in WO2016122701 DR5 VL 4012 SEQ ID NO. 94 in WO2016122701 DR5 VL 4013 SEQ ID NO. 29 in WO2016122701 DR5(Conatumumab) VL 4014 SEQ ID NO. 62 in WO2016122701 DR5(Drozitumab) VL 4015 SEQ ID NO. 54 in WO2016122701 DR5(Tigatumumab) VL 4016 SEQ ID NO. 70 in WO2016122701 E7MC VL 4017 SEQ ID NO: 238 in WO2016182957A1 E7MC VL 4018 SEQ ID NO: 239 in WO2016182957A1 E7MC VL 4019 SEQ ID NO: 240 in WO2016182957A1 E7MC VL 4020 SEQ ID NO: 241 in WO2016182957A1 E7MC VL 4021 SEQ ID NO: 242 in WO2016182957A1 E7MC VL 4022 SEQ ID NO: 243 in WO2016182957A1 E7MC VL 4023 SEQ ID NO: 36 in WO2016182957A1 E7MC VL 4024 SEQ ID NO: 37 in WO2016182957A1 E7MC VL 4025 SEQ ID NO: 38 in WO2016182957A1 E7MC VL 4026 SEQ ID NO: 39 in WO2016182957A1 E7MC VL 4027 SEQ ID NO: 41 in WO2016182957A1 E7MC VL 4028 SEQ ID NO: 42 in WO2016182957A1 E7MC VL 4029 SEQ ID NO: 43 in WO2016182957A1 E7MC VL 4030 SEQ ID NO: 44 in WO2016182957A1 E7MC VL 4031 SEQ ID NO: 45 in WO2016182957A1 E7MC VL 4032 SEQ ID NO: 46 in WO2016182957A1 E7MC VL 4033 SEQ ID NO: 47 in WO2016182957A1 E7MC VL 4034 SEQ ID NO: 48 in WO2016182957A1 E7MC VL 4035 SEQ ID NO: 49 in WO2016182957A1 E7MC VL 4036 SEQ ID NO: 50 in WO2016182957A1 E7MC VL 4037 SEQ ID NO: 51 in WO2016182957A1 E7MC VL 4038 SEQ ID NO: 52 in WO2016182957A1 E7MC VL 4039 SEQ ID NO: 53 in WO2016182957A1 E7MC VL 4040 SEQ ID NO: 54 in WO2016182957A1 E7MC VL 4041 SEQ ID NO: 55 in WO2016182957A1 E7MC VL 4042 SEQ ID NO: 56 in WO2016182957A1 EFNA VL 4043 SEQ ID NO: 151 in WO2012118547 EFNA VL 4044 SEQ ID NO: 155 in WO2012118547 EFNA VL 4045 SEQ ID NO: 159 in WO2012118547 EFNA VL 4046 SEQ ID NO: 163 in WO2012118547 EFNA4 VL 4047 SEQ ID NO. 27 in US20150125472 EFNA4 VL 4048 SEQ ID NO. 53 in US20150125472 EGFR VL 4049 SEQ ID NO. 15 in WO2015143382 EGFR VL 4050 SEQ ID NO. 14 in WO2014143765 EGFR VL 4051 EGFR VL 4052 EGFR VL 4053 EGFR VL 4054 EGFR VL 4055 EGFR VL 4056 EGFR VL 4057 EGFR VL 4058 EGFR VL 4059 EGFR VL 4060 EGFR VL 4061 EGFR VL 4062 EGFR VL 4063 EGFR VL 4064 EGFR VL 4065 EGFR VL 4066 EGFR VL 4067 EGFR VL 4068 EGFR VL 4069 EGFR VL 4070 EGFR VL 4071 EGFR VL 4072 EGFR VL 4073 EGFR VL 4074 EGFR VL 4075 EGFR VL 4076 EGFR VL 4077 EGFR VL 4078 EGFR VL 4079 EGFR VL 4080 EGFR VL 4081 EGFR VL 4082 EGFR VL 4083 EGFR VL 4084 EGFR VL 4085 EGFR VL 4086 EGFR VL 4087 EGFR VL 4088 EGFR VL 4089 EGFR VL 4090 EGFR VL 4091 EGFR VL 4092 EGFR VL 4093 EGFR VL 4094 EGFR VL 4095 EGFR VL 4096 EGFR VL 4097 EGFR VL 4098 EGFR VL 4099 EGFR VL 4100 EGFR VL 4101 EGFR VL 4102 EGFR VL 4103 EGFR VL 4104 EGFR VL 4105 EGFR VL 4106 EGFR VL 4107 EGFR VL 4108 EGFR VL 4109 EGFR VL 4110 EGFR VL 4111 EGFR VL 4112 EGFR VL 4113 EGFR VL 4114 EGFR VL 4115 EGFR VL 4116 EGFR VL 4117 EGFR VL 4118 EGFR VL 4119 EGFR VL 4120 EGFR VL 4121 EGFR VL 4122 EGFR VL 4123 EGFR VL 4124 EGFR VL 4125 EGFR VL 4126 EGFR VL 4127 EGFR VL 4128 EGFR VL 4129 EGFR VL 4130 EGFR VL 4131 EGFR VL 4132 EGFR VL 4133 EGFR VL 4134 EGFR VL 4135 EGFR VL 4136 EGFR VL 4137 EGFR VL 4138 EGFR VL 4139 EGFR VL 4140 EGFR VL 4141 EGFR VL 4142 EGFR VL 4143 EGFR VL 4144 EGFR VL 4145 EGFR VL 4146 EGFR VL 4147 EGFR VL 4148 EGFR VL 4149 EGFR VL 4150 EGFR VL 4151 EGFR VL 4152 EGFR VL 4153 EGFR VL 4154 EGFR VL 4155 EGFR VL 4156 EGFR VL 4157 EGFR VL 4158 EGFR VL 4159 EGFR VL 4160 EGFR VL 4161 EGFR VL 4162 EGFR VL 4163 EGFR VL 4164 EGFR VL 4165 EGFR VL 4166 EGFR VL 4167 EGFR VL 4168 EGFR VL 4169 EGFR VL 4170 EGFR VL 4171 EGFR VL 4172 EGFR VL 4173 EGFR VL 4174 EGFR VL 4175 EGFR VL 4176 EGFR VL 4177 EGFR VL 4178 EGFR VL 4179 EGFR VL 4180 EGFR VL 4181 EGFR VL 4182 EGFR VL 4183 EGFR VL 4184 EGFR VL 4185 EGFR VL 4186 EGFR VL 4187 EGFR VL 4188 EGFR VL 4189 EGFR VL 4190 EGFR VL 4191 EGFR VL 4192 EGFR VL 4193 EGFR VL 4194 EGFR VL 4195 EGFR VL 4196 EGFR VL 4197 EGFR VL 4198 EGFR VL 4199 EGFR VL 4200 EGFR VL 4201 EGFR VL 4202 EGFR VL 4203 EGFR VL 4204 EGFR VL 4205 EGFR VL 4206 EGFR VL 4207 EGFR VL 4208 EGFR VL 4209 EGFR VL 4210 EGFR VL 4211 EGFR VL 4212 EGFR VL 4213 EGFR VL 4214 EGFR VL 4215 EGFR VL 4216 EGFR VL 4217 EGFR VL 4218 EGFR VL 4219 EGFR VL 4220 EGFR VL 4221 EGFR VL 4222 EGFR VL 4223 EGFR VL 4224 EGFR VL 4225 EGFR VL 4226 EGFR VL 4227 EGFR VL 4228 EGFR VL 4229 EGFR VL 4230 EGFR VL 4231 EGFR VL 4232 EGFR VL 4233 EGFR VL 4234 EGFR VL 4235 EGFR VL 4236 EGFR VL 4237 EGFR VL 4238 EGFR VL 4239 EGFR VL 4240 EGFR VL 4241 EGFR VL 4242 EGFR VL 4243 EGFR VL 4244 EGFR VL 4245 EGFR VL 4246 EGFR VL 4247 EGFR VL 4248 EGFR VL 4249 EGFR VL 4250 EGFR VL 4251 EGFR VL 4252 EGFR VL 4253 EGFR VL 4254 EGFR VL 4255 EGFR VL 4256 EGFR VL 4257 EGFR VL 4258 EGFR VL 4259 EGFR VL 4260 EGFR VL 4261 EGFR VL 4262 EGFR VL 4263 EGFR VL 4264 EGFR VL 4265 EGFR VL 4266 EGFR VL 4267 EGFR VL 4268 EGFR VL 4269 EGFR VL 4270 EGFR VL 4271 EGFR VL 4272 EGFR VL 4273 EGFR VL 4274 EGFR VL 4275 EGFR VL 4276 EGFR VL 4277 EGFR VL 4278 EGFR VL 4279 EGFR VL 4280 EGFR VL 4281 EGFR VL 4282 EGFR VL 4283 EGFR VL 4284 EGFR VL 4285 EGFR VL 4286 EGFR VL 4287 EGFR VL 4288 EGFR VL 4289 EGFR VL 4290 EGFR VL 4291 EGFR VL 4292 EGFR VL 4293 EGFR VL 4294 EGFR VL 4295 EGFR VL 4296 EGFR VL 4297 EGFR VL 4298 EGFR VL 4299 EGFR VL 4300 EGFR VL 4301 EGFR VL 4302 EGFR VL 4303 EGFR VL 4304 EGFR VL 4305 EGFR VL 4306 EGFR VL 4307 EGFR VL 4308 EGFR VL 4309 EGFR VL 4310 EGFR VL 4311 EGFR VL 4312 EGFR VL 4313 EGFR VL 4314 EGFR VL 4315 EGFR VL 4316 EGFR VL 4317 EGFR VL 4318 EGFR VL 4319 EGFR VL 4320 EGFR VL 4321 EGFR VL 4322 EGFR VL 4323 EGFR VL 4324 EGFR VL 4325 EGFR VL 4326 EGFR VL 4327 EGFR VL 4328 EGFR VL 4329 EGFR VL 4330 EGFR VL 4331 EGFR VL 4332 EGFR VL 4333 EGFR VL 4334 EGFR VL 4335 EGFR VL 4336 EGFR VL 4337 EGFR VL 4338 EGFR VL 4339 EGFR VL 4340 EGFR VL 4341 EGFR VL 4342 EGFR VL 4343 EGFR VL 4344 EGFR VL 4345 EGFR VL 4346 EGFR VL 4347 EGFR VL 4348 EGFR VL 4349 EGFR VL 4350 EGFR VL 4351 EGFR VL 4352 EGFR VL 4353 EGFR VL 4354 EGFR VL 4355 EGFR VL 4356 EGFR VL 4357 EGFR VL 4358 EGFR VL 4359 EGFR VL 4360 EGFR VL 4361 EGFR VL 4362 EGFR VL 4363 EGFR VL 4364 EGFR VL 4365 EGFR VL 4366 EGFR VL 4367 EGFR VL 4368 EGFR VL 4369 EGFR VL 4370 EGFR VL 4371 EGFR VL 4372 EGFR VL 4373 EGFR VL 4374 EGFR VL 4375 EGFR VL 4376 EGFR VL 4377 EGFR VL 4378 EGFR VL 4379 EGFR VL 4380 EGFR VL 4381 EGFR VL 4382 EGFR VL 4383 EGFR VL 4384 EGFR VL 4385 EGFR VL 4386 EGFR VL 4387 EGFR VL 4388 EGFR VL 4389 EGFR VL 4390 EGFR VL 4391 EGFR VL 4392 EGFR VL 4393 EGFR VL 4394 EGFR VL 4395 EGFR VL 4396 EGFR VL 4397 EGFR VL 4398 EGFR VL 4399 EGFR VL 4400 EGFR VL 4401 EGFR VL 4402 EGFR VL 4403 EGFR VL 4404 EGFR VL 4405 EGFR VL 4406 EGFR VL 4407 EGFR VL 4408 EGFR VL 4409 EGFR VL 4410 EGFR VL 4411 EGFR VL 4412 EGFR VL 4413 EGFR VL 4414 EGFR VL 4415 EGFR VL 4416 EGFR VL 4417 EGFR VL 4418 EGFR VL 4419 EGFR VL 4420 EGFR VL 4421 EGFR VL 4422 EGFR VL 4423 EGFR VL 4424 EGFR VL 4425 EGFR VL 4426 EGFR VL 4427 EGFR VL 4428 EGFR VL 4429 EGFR VL 4430 EGFR VL 4431 EGFR VL 4432 EGFR VL 4433 EGFR VL 4434 EGFR VL 4435 EGFR VL 4436 EGFR VL 4437 EGFR VL 4438 EGFR VL 4439 EGFR VL 4440 EGFR VL 4441 EGFR VL 4442 EGFR VL 4443 EGFR VL 4444 EGFR VL 4445 EGFR VL 4446 EGFR VL 4447 EGFR VL 4448 EGFR VL 4449 EGFR VL 4450 EGFR VL 4451 EGFR VL 4452 EGFR VL 4453 EGFR VL 4454 EGFR VL 4455 EGFR VL 4456 EGFR VL 4457 EGFR VL 4458 EGFR VL 4459 EGFR VL 4460 EGFR VL 4461 EGFR VL 4462 EGFR VL 4463 EGFR VL 4464 EGFR VL 4465 EGFR VL 4466 EGFR VL 4467 EGFR VL 4468 EGFR VL 4469 EGFR VL 4470 EGFR VL 4471 EGFR VL 4472 EGFR VL 4473 EGFR VL 4474 EGFR VL 4475 EGFR VL 4476 EGFR VL 4477 EGFR VL 4478 EGFR VL 4479 EGFR VL 4480 EGFR VL 4481 EGFR VL 4482 EGFR VL 4483 EGFR VL 4484 EGFR VL 4485 EGFR VL 4486 EGFR VL 4487 EGFR VL 4488 EGFR VL 4489 EGFR VL 4490 EGFR VL 4491 EGFR VL 4492 EGFR VL 4493 EGFR VL 4494 EGFR VL 4495 EGFR VL 4496 EGFR VL 4497 EGFR VL 4498 EGFR VL 4499 EGFR VL 4500 EGFR VL 4501 EGFR VL 4502 EGFR VL 4503 EGFR VL 4504 EGFR VL 4505 EGFR VL 4506 EGFR VL 4507 EGFR VL 4508 EGFR VL 4509 EGFR VL 4510 EGFR VL 4511 EGFR VL 4512 EGFR VL 4513 EGFR VL 4514 EGFR VL 4515 EGFR VL 4516 EGFR VL 4517 EGFR VL 4518 EGFR VL 4519 EGFR VL 4520 EGFR VL 4521 EGFR VL 4522 EGFR VL 4523 EGFR VL 4524 EGFR VL 4525 EGFR VL 4526 EGFR VL 4527 EGFR VL 4528 EGFR VL 4529 EGFR VL 4530 EGFR VL 4531 EGFR VL 4532 EGFR VL 4533 EGFR VL 4534 EGFR VL 4535 EGFR VL 4536 EGFR VL 4537 EGFR VL 4538 EGFR VL 4539 EGFR VL 4540 EGFR VL 4541 EGFR VL 4542 EGFR VL 4543 EGFR VL 4544 EGFR VL 4545 EGFR VL 4546 EGFR VL 4547 EGFR VL 4548 EGFR VL 4549 EGFR VL 4550 EGFR VL 4551 EGFR VL 4552 EGFR VL 4553 EGFR VL 4554 EGFR VL 4555 EGFR VL 4556 EGFR VL 4557 EGFR VL 4558 EGFR VL 4559 EGFR VL 4560 EGFR VL 4561 EGFR VL 4562 EGFR VL 4563 EGFR VL 4564 EGFR VL 4565 EGFR VL 4566 EGFR VL 4567 EGFR VL 4568 EGFR VL 4569 EGFR VL 4570 EGFR VL 4571 EGFR VL 4572 EGFR VL 4573 EGFR VL 4574 EGFR VL 4575 EGFR VL 4576 EGFR VL 4577 EGFR VL 4578 EGFR VL 4579 EGFR VL 4580 EGFR VL 4581 EGFR VL 4582 EGFR VL 4583 EGFR VL 4584 EGFR VL 4585 EGFR VL 4586 EGFR VL 4587 EGFR VL 4588 EGFR VL 4589 EGFR VL 4590 EGFR VL 4591 EGFR VL 4592 EGFR VL 4593 EGFR VL 4594 EGFR VL 4595 EGFR VL 4596 EGFR VL 4597 EGFR VL 4598 EGFR VL 4599 EGFR VL 4600 EGFR VL 4601 EGFR VL 4602 EGFR VL 4603 EGFR VL 4604 EGFR VL 4605 EGFR VL 4606 EGFR VL 4607 EGFR VL 4608 EGFR VL 4609 EGFR VL 4610 EGFR VL 4611 EGFR VL 4612 EGFR VL 4613 EGFR VL 4614 EGFR VL 4615 EGFR VL 4616 EGFR VL 4617 EGFR VL 4618 EGFR VL 4619 EGFR VL 4620 EGFR VL 4621 EGFR VL 4622 EGFR VL 4623 EGFR VL 4624 EGFR VL 4625 EGFR VL 4626 EGFR VL 4627 EGFR VL 4628 EGFR VL 4629 EGFR VL 4630 EGFR VL 4631 EGFR VL 4632 EGFR VL 4633 EGFR VL 4634 EGFR VL 4635 EGFR VL 4636 EGFR VL 4637 EGFR VL 4638 EGFR VL 4639 EGFR(EGFRvIII) VL 4640 EGFR(EGFRvIII) VL 4641 EGFR(EGFRvIII) VL 4642 EGFR(EGFRvIII) VL 4643 EGFR(EGFRvIII) VL 4644 EGFR(EGFRvIII) VL 4645 EGFR(EGFRvIII) VL 4646 EGFR(EGFRvIII) VL 4647 EGFR(EGFRvIII) VL 4648 EGFR(EGFRvIII) VL 4649 EGFR(EGFRvIII) VL 4650 EGFR(EGFRvIII) VL 4651 EGFR(EGFRvIII) VL 4652 EGFR(EGFRvIII) VL 4653 EGFR(EGFRvIII) VL 4654 EGFR(EGFRvIII) VL 4655 EGFR(EGFRvIII) VL 4656 EGFR(EGFRvIII) VL 4657 EGFR(EGFRvIII) VL 4658 EGFRvIII VL 4659 SEQ ID NO. 14 in WO2016016341 EGFRvIII VL 4660 SEQ ID NO: 23 in WO2016168773A3 EGFRvIII VL 4661 SEQ ID NO. 42 in US20160304615 EGFRvIII VL 4662 SEQ ID NO: 1 in US20160200819A1 Endoglin VL 4663 SEQ ID NO 103 in WO2011041441 Endoglin VL 4664 SEQ ID NO 88 in WO2011041441 Endoglin VL 4665 SEQ ID NO 89 in WO2011041441 Endoglin VL 4666 SEQ ID NO 90 in WO2011041441 Endoglin VL 4667 SEQ ID NO 91 in WO2011041441 Endoglin VL 4668 SEQ ID NO 92 in WO2011041441 Endoglin VL 4669 SEQ ID NO 93 in WO2011041441 Endoglin VL 4670 SEQ ID NO 94 in WO2011041441 Endoglin VL 4671 SEQ ID NO 95 in WO2011041441 Endoglin VL 4672 SEQ ID NO 96 in WO2011041441 Endoglin VL 4673 SEQ ID NO 97 in WO2011041441 Endoglin VL 4674 SEQ ID NO. 102 in WO2011041441 Endoglin VL 4675 SEQ ID NO. 100 in WO2011041441 Endoglin VL 4676 SEQ ID NO. 100 in US20160009811 Endoglin VL 4677 SEQ ID NO. 102 in US20160009811 Endoglin VL 4678 SEQ ID NO. 103 in US20160009811 Endoglin VL 4679 SEQ ID NO. 3 in US20160009811 Endoglin VL 4680 SEQ ID NO. 4 in US20160009811 Endoglin VL 4681 SEQ ID NO. 5 in US20160009811 Endoglin VL 4682 SEQ ID NO. 70 in US20160009811 Endoglin VL 4683 SEQ ID NO. 72 in US20160009811 Endoglin VL 4684 SEQ ID NO. 74 in US20160009811 Endoglin VL 4685 SEQ ID NO. 93 in US20160009811 Endoglin VL 4686 SEQ ID NO. 94 in US20160009811 Endoglin VL 4687 SEQ ID NO. 95 in US20160009811 Endoglin VL 4688 SEQ ID NO. 96 in US20160009811 Endoglin VL 4689 SEQ ID NO. 97 in US20160009811 EphA2receptor VL 4690 US20150274824 SEQ ID NO: 26 EphA2receptor VL 4691 US20150274824 SEQ ID NO: 28 EphA2receptor VL 4692 US20150274824 SEQ ID NO: 30 EphA2receptor VL 4693 US20150274824 SEQ ID NO: 47 EphA2receptor VL 4694 US20150274824 SEQ ID NO: 48 EphA2receptor VL 4695 US20150274824 SEQ ID NO: 49 EphA2receptor VL 4696 US20150274824 SEQ ID NO: 50 EphA2receptor VL 4697 US20150274824 SEQ ID NO: 52 EphA2receptor VL 4698 US20150274824 SEQ ID NO: 78 EphA2receptor VL 4699 US20150274824 SEQ ID NO: 80 ERBB2 VL 4700 US20110129464 SEQ ID NO: 1 ERBB2 VL 4701 US20130089544 SEQ ID NO: 12 ERBB2 VL 4702 US20130089544 SEQ ID NO: 16 ERBB2 VL 4703 US20130089544 SEQ ID NO: 20 ERBB2 VL 4704 US20130089544 SEQ ID NO: 24 ERBB2 VL 4705 US20130089544 SEQ ID NO: 32 ERBB2 VL 4706 US20130089544 SEQ ID NO: 36 ERBB2 VL 4707 US20130089544 SEQ ID NO: 44 ERBB2 VL 4708 US20130089544 SEQ ID NO: 50 ERBB2 VL 4709 US20130089544 SEQ ID NO: 51 ERBB2 VL 4710 US20130089544 SEQ ID NO: 53 ERBB2 VL 4711 US20130089544 SEQ ID NO: 8 ERBB2 VL 4712 US20130266564 SEQ ID NO: 7 FactorD VL 4713 SEQ ID NO. 16 in US20160017052 FactorD VL 4714 SEQ ID NO. 18 in US20160017052 FactorD VL 4715 SEQ ID NO. 19 in US20160017052 FactorD VL 4716 SEQ ID NO. 26 in US20160017052 FactorD VL 4717 SEQ ID NO. 3 in US20160017052 FactorXII VL 4718 SEQ ID NO. 17 in WO2014089493 FAP VL 4719 SEQ ID NO. 2 in WO2015118030 FAP VL 4720 SEQ ID NO. 6 in WO2015118030 FAP VL 4721 SEQ ID NO. 171 in WO2016120216 FAP VL 4722 SEQ ID NO. 173 in WO2016120216 FAP VL 4723 SEQ ID NO: 9 in US20160326265A1 FcRL5(FcReceptorLike5) VL 4724 SEQ ID NO: 11 WO2016090337 FcRL5(FcReceptorLike5) VL 4725 SEQ ID NO: 15 WO2016090337 FcRL5(FcReceptorLike5) VL 4726 SEQ ID NO: 19 WO2016090337 FcRL5(FcReceptorLike5) VL 4727 SEQ ID NO: 23 WO2016090337 FcRL5(FcReceptorLike5) VL 4728 SEQ ID NO: 27 WO2016090337 FcRL5(FcReceptorLike5) VL 4729 SEQ ID NO: 3 WO2016090337 FcRL5(FcReceptorLike5) VL 4730 SEQ ID NO: 31 WO2016090337 FcRL5(FcReceptorLike5) VL 4731 SEQ ID NO: 35 WO2016090337 FcRL5(FcReceptorLike5) VL 4732 SEQ ID NO: 39 WO2016090337 FcRL5(FcReceptorLike5) VL 4733 SEQ ID NO: 43 WO2016090337 FcRL5(FcReceptorLike5) VL 4734 SEQ ID NO: 47 WO2016090337 FcRL5(FcReceptorLike5) VL 4735 SEQ ID NO: 7 WO2016090337 FcRL5(FcReceptorLike5) VL 4736 SEQ ID NO: 917 WO2016090337 FcRL5(FcReceptorLike5) VL 4737 SEQ ID NO: 921 WO2016090337 FGFR3 VL 4738 SEQ ID NO. 133 in U.S. Pat. No. 9,499,623 FGFR3 VL 4739 SEQ ID NO. 135 in U.S. Pat. No. 9,499,623 FGFR3 VL 4740 SEQ ID NO. 137 in U.S. Pat. No. 9,499,623 FGFR3 VL 4741 SEQ ID NO. 139 in U.S. Pat. No. 9,499,623 Frizzled Receptor VL 4742 SEQ ID NO. 12 in WO2010037041 Frizzled Receptor VL 4743 SEQ ID NO. 14 in WO2010037041 GAH VL 4744 SEQ ID NO 8 in US20060057147A1 GCC1 VL 4745 SEQ ID NO. 4 in US20160030595A1 GCC1 VL 4746 SEQ ID NO. 2 in US20160030595A1 GD2 VL 4747 SEQ ID NO. 10 in US20130216528 GD2 VL 4748 SEQ ID NO. 11 in WO2015132604 GD2 VL 4749 SEQ ID NO. 12 in WO2015132604 GD2 VL 4750 SEQ ID NO. 18 in WO2016134284 GD2 VL 4751 SEQ ID NO. 2 in US20130216528 GD2 VL 4752 SEQ ID NO. 5 in US20130216528 GD2 VL 4753 SEQ ID NO. 7 in US20130216528 GD2 VL 4754 SEQ ID NO. 9 in US20130216528 GD3 VL 4755 SEQ ID NO: 12 in WO2016185035A1 GD3 VL 4756 SEQ ID NO: 14 in WO2016185035A1 GD3 VL 4757 SEQ ID NO: 16 in WO2016185035A1 GD3 VL 4758 SEQ ID NO: 18 in WO2016185035A1 Glycol epitope and ErbB Bi VL 4759 SEQ ID No. 10 in WO2012007167A1 Specific GM2 VL 4760 US20090028877 SEQ ID NO: 21 GM2 VL 4761 US20090028877 SEQ ID NO: 24 GM2 VL 4762 US20090028877 SEQ ID NO: 25 GM2 VL 4763 US20090028877 SEQ ID NO: 31 GM2 VL 4764 US20090028877 SEQ ID NO: 32 GM2 VL 4765 US20090028877 SEQ ID NO: 33 GM2 VL 4766 US20090028877 SEQ ID NO: 34 GM2 VL 4767 US20090028877 SEQ ID NO: 35 GPC3 VL 4768 SEQ ID NO: 10 in U.S. Pat. No. 9,409,994B2 GPC3 VL 4769 SEQ ID NO: 14 in U.S. Pat. No. 9,409,994B2 GPC3 VL 4770 SEQ ID NO: 16 in US20160208015A1 GPC3 VL 4771 SEQ ID NO: 18 in U.S. Pat. No. 9,409,994B2 GPC3 VL 4772 SEQ ID NO: 22 in U.S. Pat. No. 9,409,994B2 GPC3 VL 4773 SEQ ID NO: 24 in U.S. Pat. No. 9,409,994B2 GPC3 VL 4774 SEQ ID NO: 26 in U.S. Pat. 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receptor VL 4925 SEQ ID NO. 6 in WO2004002431 Lymphotoxin beta receptor VL 4926 SEQ ID NO. 8 in WO2004002431 Lysyloxidaselike2 VL 4927 SEQ ID NO. 43 in WO2011097513 Lysyloxidaselike2 VL 4928 SEQ ID NO. 45 in WO2011097513 MCAM VL 4929 SEQ ID NO. 109 in US20150259419 MCAM VL 4930 SEQ ID NO. 110 in US20150259419 MCAM VL 4931 SEQ ID NO. 111 in US20150259419 MCAM VL 4932 SEQ ID NO. 112 in US20150259419 MCAM VL 4933 SEQ ID NO. 121 in US20150259419 MCAM VL 4934 SEQ ID NO. 122 in US20150259419 MCAM VL 4935 SEQ ID NO. 123 in US20150259419 MCAM VL 4936 SEQ ID NO. 30 in US20150239980 MCAM VL 4937 SEQ ID NO. 40 in US20150239980 MCAM VL 4938 SEQ ID NO. 50 in US20150239980 MCAM VL 4939 SEQ ID NO. 60 in US20150239980 MCAM VL 4940 SEQ ID NO. 70 in US20150239980 MCAM VL 4941 SEQ ID NO. 71 in US20150239980 MCAM VL 4942 SEQ ID NO. 72 in US20150239980 MCSF VL 4943 SEQ ID NO: 8 in WO2005030124 MCSF VL 4944 SEQ ID NO 32 in WO2005030124 MCSF VL 4945 SEQ ID NO 52 in WO2005030124 MCSF VL 4946 SEQ ID NO 60 in 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No. 8,748,585 OX40 VL 5109 SEQ ID NO. 94 in WO2016196228 OX40 VL 5110 SEQ ID NO. 98 in WO2016196228 PD1 VL 5111 SEQ ID NO. 2 in US 20160159905 PD1 VL 5112 SEQ ID NO. 21 in US20150290316 PD1 VL 5113 SEQ ID NO. 30 in US20130291136 PD1 VL 5114 SEQ ID NO. 31 in US20130291136 PD1 VL 5115 SEQ ID NO. 32 in US20130291136 PD1 VL 5116 SEQ ID NO. 33 in US20130291136 PD1 VL 5117 SEQ ID NO. 39 in US 20160159905 PD1 VL 5118 SEQ ID NO. 42 in WO2015112900 PD1 VL 5119 SEQ ID NO. 46 in WO2015112900 PD1 VL 5120 SEQ ID NO. 54 in WO2015112900 PD1 VL 5121 SEQ ID NO. 58 in WO2015112900 PD1 VL 5122 SEQ ID NO. 62 in WO2015112900 PD1 VL 5123 SEQ ID NO. 66 in WO2015112900 PD1 VL 5124 SEQ ID NO. 7 in US 20160159905 PD1 VL 5125 SEQ ID NO. 70 in WO2015112900 PD1 VL 5126 SEQ ID NO. 74 in WO2015112900 PD1 VL 5127 SEQ ID NO. 78 in WO2015112900 PD1 VL 5128 SEQ ID NO. 8 in US 20160159905 PD1 VL 5129 SEQ ID NO. 9 in US 20160159905 PD1 VL 5130 SEQ ID NO. 18 in WO2014055648 PD1(Nivolumab) VL 5131 SEQ ID NO. 11 in US20150190506 PD1(Pembrolizumab) VL 5132 SEQ ID NO. 5 in WO2016040892 PD1(Pembrolizumab) VL 5133 SEQ ID NO. 13 in US20150190506 PDK1 VL 5134 SEQ ID NO. 9 in WO2016090365 PDL1 VL 5135 SEQ ID NO. 22 in WO2016061142 PDL1 VL 5136 SEQ ID NO. 26 in WO2016061142 PDL1 VL 5137 SEQ ID NO. 30 in US20150190506 PDL1 VL 5138 SEQ ID NO. 34 in WO2016061142 PDL1 VL 5139 SEQ ID NO. 42 in WO2016061142 PDL1 VL 5140 SEQ ID NO. 58 in WO2016061142 PDL1 VL 5141 SEQ ID NO. 66 in WO2016061142 PDL1 VL 5142 SEQ ID NO. 7 in US20160319022 PDL1 VL 5143 SEQ ID NO. 74 in WO2016061142 PDL1 VL 5144 SEQ ID NO. 8 in US20150190506 PDL1 VL 5145 SEQ ID NO. 82 in WO2016061142 PDL1 VL 5146 SEQ ID NO. 86 in WO2016061142 PDL1 VL 5147 SEQ ID NO. 9 in US20150190506 PDL1 VL 5148 SEQ ID NO. 9 in US20160319022 PDL1 VL 5149 US20160108123 SEQ ID NO: 17 PDL1 VL 5150 US20160108123 SEQ ID NO: 22 PDL1 VL 5151 US20160108123 SEQ ID NO: 24 PDL1 VL 5152 US20160108123 SEQ ID NO: 249 PDL1 VL 5153 US20160108123 SEQ ID NO: 26 PDL1 VL 5154 US20160108123 SEQ ID NO: 28 PDL1 VL 5155 US20160108123 SEQ ID NO: 309 PDL1 VL 5156 US20160108123 SEQ ID NO: 311 PDL1 VL 5157 US20160108123 SEQ ID NO: 313 PDL1 VL 5158 US20160108123 SEQ ID NO: 320 PDL1 VL 5159 US20160108123 SEQ ID NO: 325 PDL1 VL 5160 US20160108123 SEQ ID NO: 34 PDL1 VL 5161 US20160108123 SEQ ID NO: 340 PDL1 VL 5162 US20160108123 SEQ ID NO: 357 PDL1 VL 5163 US20160108123 SEQ ID NO: 359 PDL1 VL 5164 US20160108123 SEQ ID NO: 36 PDL1 VL 5165 US20160108123 SEQ ID NO: 42 PDL1 VL 5166 US20160108123 SEQ ID NO: 44 PDL1 VL 5167 US20160108123 SEQ ID NO: 58 PDL1 VL 5168 US20160108123 SEQ ID NO: 60 PDL1 VL 5169 US20160108123 SEQ ID NO: 66 PDL1 VL 5170 US20160108123 SEQ ID NO: 68 PDL1 VL 5171 US20160108123 SEQ ID NO: 74 PDL1 VL 5172 US20160108123 SEQ ID NO: 76 PDL1 VL 5173 US20160108123 SEQ ID NO: 8 PDL1 VL 5174 US20160108123 SEQ ID NO: 82 PDL1 VL 5175 US20160108123 SEQ ID NO: 84 PDL1 VL 5176 US20160108123 SEQ ID NO: 86 PDL1 VL 5177 US20160108123 SEQ ID NO: 88 PDL2 VL 5178 SEQ ID NO. 47 in US20130291136 PDL2 VL 5179 SEQ ID NO. 48 in US20130291136 PDL2 VL 5180 SEQ ID NO. 49 in US20130291136 PDL2 VL 5181 SEQ ID NO. 50 in US20130291136 PDL2 VL 5182 SEQ ID NO. 51 in US20130291136 PG16 VL 5183 SEQ ID NO: 12 in EP3074419A2 PG9 VL 5184 SEQ ID NO: 10 in EP3074419A2 PGT2 VL 5185 SEQ ID NO: 16 in EP3074419A2 PGT3 VL 5186 SEQ ID NO: 18 in EP3074419A2 PGT4 VL 5187 SEQ ID NO: 20 in EP3074419A2 PGT5 VL 5188 SEQ ID NO: 22 in EP3074419A2 PRAME VL 5189 SEQ ID NO: 49 in WO2016191246A2 PRAME VL 5190 SEQ ID NO: 51 in WO2016191246A2 PRAME VL 5191 SEQ ID NO: 53 in WO2016191246A2 PRAME VL 5192 SEQ ID NO: 55 in WO2016191246A2 PRAME VL 5193 SEQ ID NO: 57 in WO2016191246A2 PRAME VL 5194 SEQ ID NO: 59 in WO2016191246A2 PRAME VL 5195 SEQ ID NO: 61 in WO2016191246A2 PRP VL 5196 SEQ ID NO: 39 in US20160333114A1 PRP VL 5197 SEQ ID NO: 41 in US20160333114A1 PSMA VL 5198 SEQ ID NO: 44 in WO2016097231 PTK7 VL 5199 SEQ ID NO. 20 in WO2012112943A1 PTK7 VL 5200 SEQ ID NO. 22 in WO2012112943A1 PTK7 VL 5201 SEQ ID NO. 24 in WO2012112943A1 PTK7 VL 5202 SEQ ID NO. 26 in WO2012112943A1 PTK7 VL 5203 SEQ ID NO. 28 in WO2012112943A1 PTK7 VL 5204 SEQ ID NO. 30 in WO2012112943A1 PTK7 VL 5205 SEQ ID NO. 32 in WO2012112943A1 PTK7 VL 5206 SEQ ID NO. 34 in WO2012112943A1 PTK7 VL 5207 SEQ ID NO. 36 in WO2012112943A1 PTK7 VL 5208 SEQ ID NO. 38 in WO2012112943A1 PTK7 VL 5209 SEQ ID NO. 40 in WO2012112943A1 PTK7 VL 5210 SEQ ID NO. 42 in WO2012112943A1 PTK7 VL 5211 SEQ ID NO. 44 in WO2012112943A1 PTK7 VL 5212 SEQ ID NO. 46 in WO2012112943A1 PTK7 VL 5213 SEQ ID NO. 48 in WO2012112943A1 PTK7 VL 5214 SEQ ID NO. 50 in WO2012112943A1 PTK7 VL 5215 SEQ ID NO. 52 in WO2012112943A1 PTK7 VL 5216 SEQ ID NO. 54 in WO2012112943A1 PTK7 VL 5217 SEQ ID NO. 56 in WO2012112943A1 PTK7 VL 5218 SEQ ID NO. 58 in WO2012112943A1 PTK7 VL 5219 SEQ ID NO. 60 in WO2012112943A1 PTK7 VL 5220 SEQ ID NO. 62 in WO2012112943A1 PTK7 VL 5221 SEQ ID NO. 64 in WO2012112943A1 PTK7 VL 5222 SEQ ID NO. 66 in WO2012112943A1 PTK7 VL 5223 SEQ ID NO. 68 in WO2012112943A1 PTK7 VL 5224 SEQ ID NO. 15 in US20150315293 PTK7 VL 5225 SEQ ID NO. 39 in US20150315293 PTK7 VL 5226 SEQ ID NO. 63 in US20150315293 RAS VL 5227 SEQ ID NO. 19 in WO2016154047 RAS VL 5228 SEQ ID NO. 49 in WO2016154047 RAS VL 5229 SEQ ID NO. 59 in WO2016154047 RAS VL 5230 SEQ ID NO. 69 in WO2016154047 RAS VL 5231 SEQ ID NO. 79 in WO2016154047 RAS VL 5232 SEQ ID NO. 9 in WO2016154047 RHAMM antagonist body VL 5233 SEQ ID NO 4 in WO2000029447 light chain Rituximab VL 5234 SEQ ID NO: 63 in US20160333114A1 Rituximab VL 5235 SEQ ID NO: 65 in US20160333114A1 ROR1 VL 5236 SEQ ID NO. 16 WO2016016343A1 ROR1 VL 5237 SEQ ID NO. 24 WO2016016343A1 ROR1 VL 5238 SEQ ID NO. 32 WO2016016343A1 ROR1 VL 5239 SEQ ID NO. 40 WO2016016343A1 ROR1 VL 5240 SEQ ID NO. 56 WO2016016343A1 ROR1 VL 5241 SEQ ID NO. 64 WO2016016343A1 ROR1 VL 5242 SEQ ID NO. 72 WO2016016343A1 ROR1 VL 5243 SEQ ID NO. 36 in WO2016016344A1 ROR1 VL 5244 SEQ ID NO. 62 in WO2016016344A1 ROR1 VL 5245 SEQ ID NO. 23 in WO2016016344A1 ROR1 VL 5246 SEQ ID NO. 49 in WO2016016344A1 ROR1 VL 5247 SEQ ID NO. 58 in WO2016016344A1 ROR1 VL 5248 SEQ ID NO. 86 in WO2016120216 ROR1 VL 5249 SEQ ID NO. 88 in WO2016120216 ROR1 VL 5250 SEQ ID NO. 90 in WO2016120216 ROR1 VL 5251 SEQ ID NO: 126 in US20160208018A1 ROR1 VL 5252 SEQ ID NO: 127 in US20160208018A1 ROR1 VL 5253 SEQ ID NO: 234 in US20160208018A1 ROR1 VL 5254 SEQ ID NO: 235 in US20160208018A1 ROR1 VL 5255 SEQ ID NO: 236 in US20160208018A1 ROR1 VL 5256 SEQ ID NO: 237 in US20160208018A1 ROR1 VL 5257 SEQ ID NO: 238 in US20160208018A1 ROR1 VL 5258 SEQ ID NO: 240 in US20160208018A1 ROR1 VL 5259 SEQ ID NO: 241 in US20160208018A1 ROR1 VL 5260 SEQ ID NO: 242 in US20160208018A1 ROR1 VL 5261 SEQ ID NO: 243 in US20160208018A1 ROR1 VL 5262 SEQ ID NO: 244 in US20160208018A1 ROR1 VL 5263 SEQ ID NO: 245 in US20160208018A1 ROR1 VL 5264 SEQ ID NO: 246 in US20160208018A1 ROR1 VL 5265 SEQ ID NO: 247 in US20160208018A1 ROR1 VL 5266 SEQ ID NO: 248 in US20160208018A1 ROR1 VL 5267 SEQ ID NO: 56 in EP3083671A1 ROR1 VL 5268 SEQ ID NO: 103 in WO2016187216A1 ROR1 VL 5269 SEQ ID NO: 111 in WO2016187216A1 ROR1 VL 5270 SEQ ID NO: 127 in WO2016187216A1 ROR1 VL 5271 SEQ ID NO: 135 in WO2016187216A1 ROR1 VL 5272 SEQ ID NO: 143 in WO2016187216A1 ROR1 VL 5273 SEQ ID NO: 15 in WO2016187216A1 ROR1 VL 5274 SEQ ID NO: 151 in WO2016187216A1 ROR1 VL 5275 SEQ ID NO: 159 in WO2016187216A1 ROR1 VL 5276 SEQ ID NO: 167 in WO2016187216A1 ROR1 VL 5277 SEQ ID NO: 175 in WO2016187216A1 ROR1 VL 5278 SEQ ID NO: 183 in WO2016187216A1 ROR1 VL 5279 SEQ ID NO: 191 in WO2016187216A1 ROR1 VL 5280 SEQ ID NO: 199 in WO2016187216A1 ROR1 VL 5281 SEQ ID NO: 207 in WO2016187216A1 ROR1 VL 5282 SEQ ID NO: 215 in WO2016187216A1 ROR1 VL 5283 SEQ ID NO: 223 in WO2016187216A1 ROR1 VL 5284 SEQ ID NO: 23 in WO2016187216A1 ROR1 VL 5285 SEQ ID NO: 231 in WO2016187216A1 ROR1 VL 5286 SEQ ID NO: 239 in WO2016187216A1 ROR1 VL 5287 SEQ ID NO: 247 in WO2016187216A1 ROR1 VL 5288 SEQ ID NO: 255 in WO2016187216A1 ROR1 VL 5289 SEQ ID NO: 263 in WO2016187216A1 ROR1 VL 5290 SEQ ID NO: 271 in WO2016187216A1 ROR1 VL 5291 SEQ ID NO: 279 in WO2016187216A1 ROR1 VL 5292 SEQ ID NO: 287 in WO2016187216A1 ROR1 VL 5293 SEQ ID NO: 295 in WO2016187216A1 ROR1 VL 5294 SEQ ID NO: 303 in WO2016187216A1 ROR1 VL 5295 SEQ ID NO: 31 in WO2016187216A1 ROR1 VL 5296 SEQ ID NO: 311 in WO2016187216A1 ROR1 VL 5297 SEQ ID NO: 319 in WO2016187216A1 ROR1 VL 5298 SEQ ID NO: 327 in WO2016187216A1 ROR1 VL 5299 SEQ ID NO: 335 in WO2016187216A1 ROR1 VL 5300 SEQ ID NO: 343 in WO2016187216A1 ROR1 VL 5301 SEQ ID NO: 351 in WO2016187216A1 ROR1 VL 5302 SEQ ID NO: 359 in WO2016187216A1 ROR1 VL 5303 SEQ ID NO: 39 in WO2016187216A1 ROR1 VL 5304 SEQ ID NO: 47 in WO2016187216A1 ROR1 VL 5305 SEQ ID NO: 55 in WO2016187216A1 ROR1 VL 5306 SEQ ID NO: 63 in WO2016187216A1 ROR1 VL 5307 SEQ ID NO: 7 in WO2016187216A1 ROR1 VL 5308 SEQ ID NO: 71 in WO2016187216A1 ROR1 VL 5309 SEQ ID NO: 79 in WO2016187216A1 ROR1 VL 5310 SEQ ID NO: 87 in WO2016187216A1 ROR1 VL 5311 SEQ ID NO: 95 in WO2016187216A1 SEMAPHORIN4D VL 5312 SEQ ID NO. 17 in US20160115240A1 SEMAPHORIN4D VL 5313 SEQ ID NO. 18 in US20160115240A1 SEMAPHORIN4D VL 5314 SEQ ID NO. 29 in US20160115240A1 TAG73 VL 5315 SEQ ID NO: 116 in US20160333114A1 TCR VL 5316 SEQ ID NO. 132 in WO2016122701 TEM8 VL 5317 SEQ ID NO: 4 in US20160264662A1 TEM8 VL 5318 SEQ ID NO: 6 in US20160264662A1 TEM8 VL 5319 SEQ ID NO: 8 in US20160264662A1 Tie VL 5320 SEQ ID NO 724 in US20060057138A1 TIGIT VL 5321 SEQ ID NO. 130 in US20160355589 TIGIT VL 5322 SEQ ID NO. 131 in US20160355589 TIGIT VL 5323 SEQ ID NO. 132 in US20160355589 TIGIT VL 5324 SEQ ID NO. 133 in US20160355589 TIGIT VL 5325 SEQ ID NO. 137 in US20160355589 TIGIT VL 5326 SEQ ID NO. 139 in US20160355589 TIGIT VL 5327 SEQ ID NO. 145 in US20160355589 TIGIT VL 5328 SEQ ID NO. 146 in US20160355589 TIGIT VL 5329 SEQ ID NO. 151 in US20160355589 TIGIT VL 5330 SEQ ID NO. 152 in US20160355589 TIGIT VL 5331 SEQ ID NO. 25 in US20160355589 TIGIT VL 5332 SEQ ID NO. 26 in US20160355589 TIGIT VL 5333 SEQ ID NO. 27 in US20160355589 TIGIT VL 5334 SEQ ID NO. 28 in US20160355589 TIGIT VL 5335 SEQ ID NO. 29 in US20160355589 TIGIT VL 5336 SEQ ID NO. 30 in US20160355589 TIGIT VL 5337 SEQ ID NO. 50 in US20160355589 TIGIT VL 5338 SEQ ID NO. 51 in US20160355589 TIGIT VL 5339 SEQ ID NO. 52 in US20160355589 TIGIT VL 5340 SEQ ID NO. 64 in US20160355589 TIGIT VL 5341 SEQ ID NO. 95 in US20160355589 TIGIT VL 5342 SEQ ID NO. 8 in US20160355589 TIM3 VL 5343 SEQ ID NO: 7 in WO2013006490 TIM3 VL 5344 SEQ ID NO. 107 in US20150086574 TIM3 VL 5345 SEQ ID NO. 117 in US20150086574 TIM3 VL 5346 SEQ ID NO. 17 in US20150086574 TIM3 VL 5347 SEQ ID NO. 27 in US20150086574 TIM3 VL 5348 SEQ ID NO. 37 in US20150086574 TIM3 VL 5349 SEQ ID NO. 47 in US20150086574 TIM3 VL 5350 SEQ ID NO. 57 in US20150086574 TIM3 VL 5351 SEQ ID NO. 67 in US20150086574 TIM3 VL 5352 SEQ ID NO. 7 in US20150086574 TIM3 VL 5353 SEQ ID NO. 77 in US20150086574 TIM3 VL 5354 SEQ ID NO. 87 in US20150086574 TIM3 VL 5355 SEQ ID NO. 97 in US20150086574 TIM3 VL 5356 SEQ ID NO: 17 in WO2016179319A1 TIM3 VL 5357 SEQ ID NO: 25 in WO2016179319A1 TIM3 VL 5358 SEQ ID NO: 33 in WO2016179319A1 TIM3 VL 5359 SEQ ID NO: 41 in WO2016179319A1 TIM3 VL 5360 SEQ ID NO: 49 in WO2016179319A1 TIM3 VL 5361 SEQ ID NO: 57 in WO2016179319A1 TIM3 VL 5362 SEQ ID NO: 65 in WO2016179319A1 TIM3 VL 5363 SEQ ID NO: 73 in WO2016179319A1 TIM3 VL 5364 SEQ ID NO: 81 in WO2016179319A1 TIM3 VL 5365 SEQ ID NO: 89 in WO2016179319A1 TIM3 VL 5366 SEQ ID NO: 9 in WO2016179319A1 TIM3 VL 5367 SEQ ID NO: 97 in WO2016179319A1 Tissue factor VL 5368 SEQ ID NO 25 in US20040229301A1 Tissue factor VL 5369 SEQ ID NO 31 in US20040229301A1 Tissue factor VL 5370 SEQ ID NO 12 in WO2004094475 Tissue factor VL 5371 SEQ ID NO 21 in WO2004094475 Tissue factor VL 5372 SEQ ID NO 25 in WO2004094475 Tissue factor VL 5373 SEQ ID NO 31 in WO2004094475 Tissue factor VL 5374 SEQ ID NO 8 in WO2004094475 Tissue factor VL 5375 SEQ ID NO: 35 in US20160333114A1 Tissue factor VL 5376 SEQ ID NO: 37 in US20160333114A1 TRBC1 VL 5377 SEQ ID NO. 2 in WO2015132598 TrophoblastGlycoprotein5T4 VL 5378 SEQ ID NO. 18 in WO2016034666A1 TrophoblastGlycoprotein5T4 VL 5379 SEQ ID NO. 12 in WO2016034666A1 VL TrophoblastGlycoprotein5T4 VL 5380 SEQ ID NO. 14 in WO2016034666A1 VL TrophoblastGlycoprotein5T4 VL 5381 SEQ ID NO. 16 in WO2016034666A1 VL uPAR VL 5382 SEQ ID NO: 71 in US20160333114A1 uPAR VL 5383 SEQ ID NO: 73 in US20160333114A1 VEGF VL 5384 SEQ ID NO 2 in WO2000034337 VEGF VL 5385 SEQ ID NO 6 in WO2000034337 VEGF VL 5386 SEQ ID NO. 9 in US20030175276A1 VEGF VL 5387 SEQ ID NO 11 in WO2006012688A1 VEGF VL 5388 SEQ ID NO 19 in WO2006012688A1 VEGF VL 5389 SEQ ID NO 27 in WO2006012688A1 VEGF VL 5390 SEQ ID NO 28 in WO2006012688A1 VEGF VL 5391 SEQ ID NO 3 in WO2006012688A1 VEGF VL 5392 SEQ ID NO 43 in WO2006012688A1 VEGF VL 5393 US20160090427 SEQ ID NO: 160 VEGF VL 5394 US20160090427 SEQ ID NO: 161 VEGF VL 5395 US20160090427 SEQ ID NO: 162 VEGF VL 5396 US20160090427 SEQ ID NO: 163 VEGF VL 5397 US20160090427 SEQ ID NO: 164 VEGF VL 5398 US20160090427 SEQ ID NO: 165 VEGF VL 5399 US20160090427 SEQ ID NO: 166 VEGF VL 5400 US20160090427 SEQ ID NO: 167 VEGFR2 VL 5401 SEQ ID NO. 107 in WO2017004254 VEGFR2 VL 5402 SEQ ID NO. 108 in WO2017004254 VEGFR2 VL 5403 SEQ ID NO. 109 in WO2017004254 VEGFR2 VL 5404 SEQ ID NO. 110 in WO2017004254 VEGFR2 VL 5405 SEQ ID NO. 111 in WO2017004254 VEGFR2 VL 5406 SEQ ID NO. 112 in WO2017004254 VEGFR2 VL 5407 SEQ ID NO. 113 in WO2017004254 VEGFR2 VL 5408 SEQ ID NO. 86 In WO2017004254 VEGFR2 VL 5409 SEQ ID NO. 87 In WO2017004254 VEGFR2 VL 5410 SEQ ID NO. 88 In WO2017004254 VEGFR2 VL 5411 SEQ ID NO. 89 In WO2017004254 VEGFR2 VL 5412 SEQ ID NO. 90 In WO2017004254 VEGFR2 VL 5413 SEQ ID NO. 91 In WO2017004254 VEGFR2 VL 5414 SEQ ID NO. 92 In WO2017004254 VEGFR2 VL 5415 SEQ ID NO. 93 In WO2017004254 VEGFR2 VL 5416 SEQ ID NO. 94 In WO2017004254 VISTA VL 5417 SEQ ID NO: 41 in WO2015097536 VISTA VL 5418 SEQ ID NO: 42 in WO2015097536 VISTA VL 5419 SEQ ID NO: 43 in WO2015097536 VISTA VL 5420 SEQ ID NO: 44 in WO2015097536 VISTA VL 5421 SEQ ID NO: 45 in WO2015097536 VMS2 VL 5422 FIG. 2 in WO2000058363 WT1/HLA Bi Specific VL 5423 SEQ ID NO. 106 in WO2015070061 WT1/HLA Bi Specific VL 5424 SEQ ID NO. 112 in WO2015070061 WT1/HLA Bi Specific VL 5425 SEQ ID NO. 130 in WO2015070061 WT1/HLA Bi Specific VL 5426 SEQ ID NO. 34 in WO2015070061 WT1/HLA Bi Specific VL 5427 SEQ ID NO. 52 in WO2015070061 WT1/HLA Bi Specific VL 5428 SEQ ID NO. 70 in WO2015070061 WT1/HLA Bi Specific VL 5429 SEQ ID NO. 88 in WO2015070061 5T4 VL 5430 SEQ ID NO. 1 in WO2016022939 5T4 VL 5431 SEQ ID NO. 3 in WO2016022939 ALK VL 5432 SEQ ID NO. 10 in WO2015069922 ALK VL 5433 SEQ ID NO. 12 in WO2015069922 ALK VL 5434 SEQ ID NO. 14 in WO2015069922 ALK VL 5435 SEQ ID NO. 16 in WO2015069922 ALK VL 5436 SEQ ID NO. 8 in WO2015069922 ALKVL VL 5437 SEQ ID NO. 2 in WO2015069922 ALKVL VL 5438 SEQ ID NO. 4 in WO2015069922 ALKVL VL 5439 SEQ ID NO. 6 in WO2015069922 CD123 VL 5440 SEQ ID NO. 114 in WO2016120216 CD123 VL 5441 SEQ ID NO. 116 in WO2016120216 CD123 VL 5442 SEQ ID NO. 58 in WO2016120216 CD123 VL 5443 SEQ ID NO. 60 in WO2016120216 CD123 VL 5444 SEQ ID NO. 64 in WO2016120216 CD16 VL 5445 SEQ ID NO. 26 in WO2015158868 CD22 VL 5446 SEQ ID NO. 1 in WO2013059593 CD276 VL 5447 SEQ ID NO. 18 in US20160053017 CD276 VL 5448 SEQ ID NO. 27 in US20160053017 CD28 VL 5449 SEQ ID NO. 20 in WO2015158868 CD73 VL 5450 SEQ ID NO. 101 in US20160145350 CD73 VL 5451 SEQ ID NO. 102 in US20160145350 CD73 VL 5452 SEQ ID NO. 104 in US20160145350 CD73 VL 5453 SEQ ID NO. 106 in US20160145350 CD73 VL 5454 SEQ ID NO. 110 in US20160145350 CD73 VL 5455 SEQ ID NO. 117 in US20160145350 CD73 VL 5456 SEQ ID NO. 118 in US20160145350 CD73 VL 5457 SEQ ID NO. 120 in US20160145350 CD73 VL 5458 SEQ ID NO. 122 in US20160145350 CD74 VL 5459 SEQ ID NO. 25 in US20130171064 CD74 VL 5460 SEQ ID NO. 29 in US20130171064 CD74 VL 5461 SEQ ID NO. 31 in US20130171064 CD74 VL 5462 SEQ ID NO. 35 in US20130171064 CS1 VL 5463 SEQ ID NO. 110 in WO2016120216 CS1 VL 5464 SEQ ID NO. 112 in WO2016120216 CSPG4 VL 5465 SEQ ID NO. 12 in WO2016077638 CSPG4 VL 5466 SEQ ID NO. 14 in WO2016077638 EGFRvIII VL 5467 SEQ ID NO. 92 in WO2016120216 EGFRvIII VL 5468 SEQ ID NO. 94 in WO2016120216 ERBB2 VL 5469 US20110129464 SEQ ID NO: 3 GPC3 VL 5470 SEQ ID NO. 23 in WO2016049459 Malignant Variable Receptor VL 5471 SEQ ID NO. 5 in WO2015133817A1 OX40 VL 5472 SEQ ID NO. 29 in US20160137740 OX40 VL 5473 SEQ ID NO. 37 in US20150190506

Intrabodies

In some embodiments, payloads of the present invention may be intrabodies against players in regulating immune cells. An intrabody is an antibody that is designed to be expressed intracellularly and can be directed to a specific target antigen present in various subcellular locations including the cytosol, nucleus, endoplasmic reticulum (ER), mitochondria, peroxisomes, plasma membrane and trans-Golgi network (TGN) through in frame fusion with intracellular trafficking/localization peptide sequences. The most commonly used format is a single chain antibody (scFv) created by joining the antigen-binding variable domains of heavy and light chain with a linker, most often the 15-amino acid linker (e.g., (GGGGS)₃ (SEQ ID NO: 307)) between the variable heavy (VH) and variable light (VL) chains. The intracellular intrabodies are being developed to bind to, neutralize, or modify the function or localization of cancer-related targets and thereby affect the malignant phenotype.

Players that modulate immune cells (e.g., T cells) may be any intracellular signaling checkpoint. Exemplary players may include a co-inhibitory ligand, Casitas B-linage lymphoma proto-oncogene-b (Cb1-b) (a E3 ligase), a protein tyrosine phosphatase (PTP) such as Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1), and Ras. For example, an intrabody may be an intrabody against oncogenic form of RAS disclosed in PCT patent publication NO.: WO2004/046186; the contents of which are incorporated by reference in their entirety.

Therapeutic Antibodies

In some embodiments, antibody payloads of the present invention may be therapeutic antibodies. As non-limiting examples, antibodies and fragments and variants thereof may be specific to tumor associated antigens, or tumor specific antigens, or pathogen antigens. In some aspects, antibodies may be blocking antibodies (also referred to as antagonistic antibodies), for example, blocking antibodies against PD-1, PD-L1, PD-L2, CTLA-4 and other inhibitory molecules. In other aspects, antibodies may be agonist antibodies such as agonistic antibodies specific to stimulatory molecules, e g., 4-1BB (CD137), OX40 (CD134), CD40, GITR and CD27.

Other exemplary therapeutic antibodies may include, but are not limited to, Abagovomab, Abcxmab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Afasevikumab, Afelimomab, Afutuzumab, Alacizumab, Alemtuzumab, Alirocumab, Altumomab, Amatuximab, Anetumab, Anifrolumab, Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Atlizumab, Atorolimumab, Avelumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab, Bleselumab, Blinatumomab, Blinatumomab, Blosozumab, Bococizumab, Brentuximab, Briaknumab, Brodalumab, Brolucizumab, Brontictuzumab, Cabiralizumab, Canakinumab, Cantuzumab, Caplacizumab, Capromab, Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Cergutuzumab, Certolizumab pegol, Cetuximab, Citatuzumab, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab, Codrituzumab, Coltuximab, Contatumumab, Concizumab, Crenezumab, Crotedumab, CR6261, Dacetumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab, Denosumab, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Domagrozumab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Emicizumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galcanezumab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tituxetan, icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab, Indusatumab, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lanadelumab, Landogrozumab, Laprituximab, Lebrikizumab, Lemalesomab, Lendalizumab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab, Ligelizumab, Lilotomab, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirvetuximab, Mitumomab, Mogamulizumab, Monalizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, nacolomab tafenatox, Namilumab, naptumomab, naratuximab, Narnatumab, Natalizumab, Navicixizumab, Navivumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab, Pintumomab, Placulumab, Plozalizumab, Pogalizumab, Polatuzumab, Ponezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Risankizumab, Rituximab, Rivabazumab pegol, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovalpituzumab, Rovelizumab, Ruplizumab, Sacituzumab, Samalizumab, Sapelizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab vedotin, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, tabalumab, Tacatuzumab, Tadocizumab, Talizumab, Tamtuvetmab, Tanezumab, Taplitumomab, Tarextumab, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Ticilimumab, Tildrakizumab, Tigatuzumab, Timolumab, Tisotumab vedotin, TNX-650, Tocilizumab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab, Tuvirumab, Ublituximab, Ulcocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Vadastuximab talirine, Vandortuzumab vedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Volociximab, Vorsetuzumab, Votumumab, Xentuzumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab and Zolimomab aritox.

Bicistronic and/or Pseudo-Bicistronic Antibody Payloads

According to the present invention, a bicistronic payload is a polynucleotide encoding a two-protein chain antibody on a single polynucleotide strand. A pseudo-bicistronic payload is a polynucleotide encoding a single chain antibody discontinuously on a single polynucleotide strand. For bicistronic payloads, the encoded two strands or two portions/regions and/or domains (as is the case with pseudo-bicistronic) are separated by at least one nucleotide not encoding the strands or domains. More often the separation comprises a cleavage signal or site or a non-coding region of nucleotides. Such cleavage sites include, for example, furin cleavage sites encoded as an “RKR” site, or a modified furin cleavage site in the resultant polypeptide or any of those taught herein.

According to the present invention, a single domain payload comprises one or two polynucleotides encoding a single monomeric variable antibody domain. Typically, single domain antibodies comprise one variable domain (VH) of a heavy-chain antibody.

According to the present invention, a single chain Fv payloads is a polynucleotide encoding at least two coding regions and a linker region. The scFv payload may encode a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N− terminus of the VH with the C-terminus of the VL, or vice versa. Other linkers include those known in the art and disclosed herein.

According to the present invention, a bispecific payload is a polynucleotide encoding portions or regions of two different antibodies. Bispecific payloads encode polypeptides which may bind two different antigens. Polynucleotides of the present invention may also encode trispecific antibodies having an affinity for three antigens.

3. Tumor and Pathogen Specific Antigens

In some embodiments, payloads of the present invention may be tumor specific antigens (TSAs), tumor associated antigens (TAAs), pathogen associated antigens, or fragments thereof. The antigen can be expressed as a peptide or as an intact protein or portion thereof. The intact protein or a portion thereof can be native or mutagenized. Antigens associated with cancers or virus-induced cancers as described herein are well-known in the art. Such a TSA or TAA may be previously associated with a cancer or may be identified by any method known in the art.

A tumor specific antigen (TSA) may be a tumor neoantigen. A neoantigen is a mutated antigen that is only expressed by tumor cells because of genetic mutations or alterations in transcription which alter protein coding sequences, therefore creating novel, foreign antigens. The genetic changes result from genetic substitution, insertion, deletion or any other genetic changes of a native cognate protein (i.e. a molecule that is expressed in normal cells).

As non-limiting examples, neoantigens may include mutated new peptides derived from alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDKN2A, CLPP, CIVIL-66, COA-1, connexin 37, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, fibronectin, FLT3-ITD, FN1, GPNM8, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, Hsp-70-1B, MART-2, MEL MUM-1, MUM-2, MUM-3, Myosin class I, NFYC, neo-PAP, OGT, OS-9, p53, pm1-RARalpha fusion protein, PRDX5, PTPRK, K-Ras, N-Ras, RBAF600, sirtuin-2, SNRPD1, SYT-SSX1/SSX2 fusion protein, TGF-beta receptor II, etc. Additional neoantigen peptides may include SF3B1 peptides, MYD peptides, TP53 peptides, Abl peptides, FBXW7 peptides, MAPK peptides, and GNB1 peptides disclosed in US patent publication NO.: 20110293637; the contents of which are incorporated herein by reference in their entirety.

New neoantigens identified through large-scale sequencing and algorithm calculation may also be included. See, e.g., International Patent Publication NO.: WO2014/168874; Nishimura et al., Cancer Sci. 2015, 106(5): 505-511; and Linnemann et al., Nat. Med., 2015, 21(1): 81-85; the contents of each of which are incorporated herein by reference in their entirety.

A tumor associated antigen (TAA) may be an overexpressed or accumulated antigen that is expressed by both normal and neoplastic tissue, with the level of expression highly elevated in cancer tissues. Numerous proteins (e.g. oncogenes) are up-regulated in tumor tissues, including but not limited to adipophilin, AIM-2, ALDH1A1, BCLX(L), BING-4, CALCA, CD45, CD274, CPSF, cyclin D1, DKK1, ENAH, epCAM, ephA3, EZH2, FGF5, G250, HER-2/neu, HLA-DOB, Hepsin, IDO1, IGFB3, IL13Ralpha2, Intestinal carboxyl esterase, kallikrein 4, KIF20A, lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC-1, MUC5AC, p53, Pax5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RU2A5, SECERNIN 1, SOX10, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1.

A TAA may be an oncofetal antigen that is typically only expressed at different stages during the development of the fetus and in cancerous somatic cells. Many proteins are normally expressed during fetal development but are transcriptionally repressed after birth or at early stage of infancy, therefore are not present, or are expressed in significantly lower levels in the corresponding normal adult tissue. Some of these developmental proteins are re-expressed in certain tumor cells and become oncofetal antigens. Examples of oncofetal antigens may include, but are not limited to CEA (carcinoembryonic antigen) in colorectal carcinoma, iLRP/OFA (immature laminin receptor protein/oncofetal antigen) in renal cell carcinoma (RCC), TAG-72 (tumor associated glycoprotein-72) in prostate carcinoma, AFP (alpha-fetoprotein) in hepatocellular carcinoma (HCC), ROR1 (a receptor tyrosine kinase) in many malignant cells such as brain tumors, sperm protein 17, HMGA2 (high mobility group A2) in ovarian carcinoma, oncofetal H19, CR-1 (Cripto-1, a member of epidermal growth factor (EGF)-CFC family), trophoblast glycoprotein precursor and GPC-3 (Glypican-3, a member of heparan sulphate proteoglycans) in HCC.

A TAA may be a cancer-testis antigen that is expressed only by cancer cells and adult reproductive tissues such as testis and placenta, including, but limited to antigens from BAGE family, CAGE family, HAGE family, GAGE family, MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6 and MAGE-A13), SAGE family, XAGE family, MCAK, NA88-A (cancer/testis antigen 88), PSAD1, SSX-2, and SLLP-1.

A TAA may be a lineage restricted antigen that is expressed largely by a single cancer histotype, such as Melan-A/MART-1, Gp100/pmel17, Tyrosinase, TRP-1/-2, P. polypeptide, MC1R in melanoma; and prostate specific antigen (PSA) in prostate cancer.

A TAA may be an oncoviral antigen that is encoded by tumorigenic transforming viruses (also called oncogenic viruses). Oncogenic viruses, when they infect host cells, can insert their own DNA (or RNA) into that of the host cells. When the viral DNA or RNA affects the host cell's genes, it can push the cell toward becoming cancer. Oncogenic viruses include, but are not limited to, RNA viruses, and DNA viruses. Some examples of commonly known oncoviruses include human papilloma viruses (HPVs) which are main causes of cervical cancer, Epstein-Barr virus (EBV) which may cause nasopharyngeal cancer, certain types of fast-growing lymphomas (e.g., Burkitt lymphoma) and stomach cancer, hepatitis B, C and D viruses (HBV, HCV and HDV) in hepatocellular carcinoma (HCC), human immunodeficiency virus (HIV) which increases the risk of getting many types of cancer (e.g., liver cancer, anal cancer and Hodgkin cancer), Kaposi sarcoma herpes virus (KSHV; also known as human herpes virus 8 (HHV8)) which is linked to lymphoma, human T-lymphotrophic virus (HTLV-1) and merkel cell polymavirus (MCV). A viral antigen can be any defined antigen of a virus that is associated with a cancer in a human. For example, antigens from EBV may include but are not limited to, Epstein-Barr nuclear antigen-1 (EBNA1), latent membrane protein 1 (LMP1), or latent membrane protein 2 (LMP2).

A TAA may be an idiotypic antigen that is generated from highly polymorphic genes where a tumor cell expresses a specific “clonotype”, i.e., as in B cell, T cell lymphoma/leukemia resulting from clonal aberrancies, such as Immunoglobulin and T cell receptors (TCRs). Idiotypic antigens are a class of non-pathogen-associated neoantigens. For example, the malignant B cells express rearranged and multiply mutated surface immunoglobulins (Ig). Tumor specific idiotypes (e.g., immunoglobulin idiotypes) are regarded as particularly attractive tumor-specific antigens that can be successfully targeted by immunotherapy (e.g., Alejandro et al., Front Oncol., 2012, 2: 159).

4. T Cell Receptors (TCRs)

In some embodiments, payloads of the present invention may be T cell receptors (TCRs). The TCR may be specific to a cancer antigen, having a specific α chain and β chain which together form a TCRαβ heterodimer, or having a specific γ chain and δ chain which together form a TCRγδ heterodimer. The TCR may be a recombinant antigen specific T cell receptor.

The variable regions of a chain and β chain determine T cell specificity to an antigenic peptide presented by the major histocompatibility complex (MEW) class I and II molecules. The TCR recognition of the tumor antigen on the surface of a tumor cell presented by MEW molecules by TCR triggers T cell activation. The use of TCR gene therapy can equip a subject's own T cells with desired specificities and generate sufficient numbers of T cells to eradicate tumor cells. In some embodiments, a biocircuit or an effector module comprising a tumor specific TCR may be transduced into T cells and TCR-engineered T cells will be infused into cancer patients who have lymphocytopenia or lymphopenia by chemotherapy or irradiation, allowing efficient engraftment but inhibiting immune suppression.

Sequences encoding tumor antigen recognizing TCR chains can be obtained from tumor-reactive T cells (e.g., tumor-infiltrating lymphocytes isolated from the tumor of a patient).

According to the present invention, a TCR specific to tumor cells can be produced by methods described in International Patent Publication NO.: WO2014/083173; the contents of which are incorporated herein by reference in their entirety. A host organism expressing a transgene of a human leucocyte antigen (HLA) type which is known or suspected to be able to present a mutated tumor specific antigen (TSA) is transduced to express the un-rearranged human TCR loci. Preferably these loci encode TCR α and β chains, and preferably comprise a plurality, ideally all, of human TCR V, D, J, and/or C genes. The host organism is immunized with a cancer specific TSA or a peptide epitope derived from the TSA and T cells expressing rearranged TCRs specifically against the TSA are isolated and cloned. The TCR from the cloned T cells are sequenced (International Patent Publication NO.: WO2014/083173; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, payloads of the present invention may be TCRs that specifically recognize TSAs, TAAs, or epitopes thereof, complexed with MEW molecules.

Exemplary tumor antigens that can be recognized by a TCR may include at least the following: 5T4, 707-AP, A33, AFP (α-fetoprotein), AKAP-4 (A kinase anchor protein 4), ALK, α5β1-integrin, androgen receptor, annexin II, alpha-actinin-4, ART-4, B1, B7H3, B7H4, BAGE (B melanoma antigen), BCMA, BCR-ABL fusion protein, beta-catenin, BKT-antigen, BTAA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calretinin, CAIX (carbonic anhydrase), CAMEL (cytotoxic T-lymphocyte recognized antigen on melanoma), CAM43, CAP-1, Caspase-8/m, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD25, CD27/m, CD28, CD30, CD33, CD34, CD36, CD38, CD40/CD154, CD41, CD44v6, CD44v7/8, CD45, CD49f, CD56, CD68\KP1, CD74, CD79a/CD79b, CD103, CD123, CD133, CD138, CD171, cdc27/m, CDK4 (cyclin dependent kinase 4), CDKN2A, CDS, CEA (carcinoembryonic antigen), CEACAM5, CEACAM6, chromogranin, c-Met, c-Myc, coa-1, CSAp, CT7, CT10, cyclophilin B, cyclin B1, cytoplasmic tyrosine kinases, cytokeratin, DAM-10, DAM-6, dek-can fusion protein, desmin, DEPDC1 (DEP domain containing 1), E2A-PRL, EBNA, EGF-R (epidermal growth factor receptor), EGP-1 (epithelial glycoprotein −1) (TROP-2), EGP-2, EGP-40, EGFR (epidermal growth factor receptor), EGFRvIII, EF-2, ELF2M, EMMPRIN, EpCAM (epithelial cell adhesion molecule), EphA2, Epstein Barr virus antigens, Erb (ErbB1; ErbB3; ErbB4), ETA (epithelial tumor antigen), ETV6-AML1 fusion protein, FAP (fibroblast activation protein), FBP (folate-binding protein), FGF-5, folate receptor a, FOS related antigen 1, fucosyl GM1, G250, GAGE (GAGE-1; GAGE-2), galactin, GD2 (ganglioside), GD3, GFAP (glial fibrillary acidic protein), GM2 (oncofetal antigen-immunogenic-1; OFA-I-1), GnT-V, Gp100, H4-RET, HAGE (helicase antigen), HER-2/neu, HIFs (hypoxia inducible factors), HIF-1α, HIF-2α, HLA-A2, HLA-A*0201-R170I, HLA-A11, HMWMAA, Hom/Mel-40, HSP70-2M (Heat shock protein 70), HST-2, HTgp-175, hTERT (or hTRT), human papillomavirus-E6/human papillomavirus-E7 and E6, iCE (immune-capture EIA), IGF-1R, IGH-IGK, IL2R, IL5, ILK (integrin-linked kinase), IMP3 (insulin-like growth factor II mRNA-binding protein 3), IRF4 (interferon regulatory factor 4), KDR (kinase insert domain receptor), KIAA0205, KRAB-zinc finger protein (KID)-3; KID31, KSA (17-1A), K-ras, LAGE, LCK, LDLR/FUT (LDLR-fucosyltransferaseAS fusion protein), LeY (Lewis Y), MAD-CT-1, MAGE (tyrosinase, melanoma-associated antigen) (MAGE-1; MAGE-3), melan-A tumor antigen (MART), MART-2/Ski, MC1R (melanocortin 1 receptor), MDM2, mesothelin, MPHOSPH1, MSA (muscle-specific actin), mTOR (mammalian targets of rapamycin), MUC-1, MUC-2, MUM-1 (melanoma associated antigen (mutated) 1), MUM-2, MUM-3, Myosin/m, MYL-RAR, NA88-A, N-acetylglucosaminyltransferase, neo-PAP, NF-KB (nuclear factor-kappa B), neurofilament, NSE (neuron-specific enolase), Notch receptors, NuMa, N-Ras, NY-BR-1, NY-CO-1, NY-ESO-1, Oncostatin M, OS-9, OY-TES1, p53 mutants, p190 minor bcr-ab1, p15(58), p185erbB2, p180erbB-3, PAGE (prostate associated gene), PAP (prostatic acid phosphatase), PAX3, PAX5, PDGFR (platelet derived growth factor receptor), cytochrome P450 involved in piperidine and pyrrolidine utilization (PIPA), Pm1-RAR alpha fusion protein, PR-3 (proteinase 3), PSA (prostate specific antigen), PSM, PSMA (Prostate stem cell antigen), PRAME (preferentially expressed antigen of melanoma), PTPRK, RAGE (renal tumor antigen), Raf (A-Raf, B-Raf and C-Raf), Ras, receptor tyrosine kinases, RCAS1, RGSS, ROR1 (receptor tyrosine kinase-like orphan receptor 1), RU1, RU2, SAGE, SART-1, SART-3, SCP-1, SDCCAG16, SP-17 (sperm protein 17), src-family, SSX (synovial sarcoma X breakpoint)-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, STAT-3, STAT-5, STAT-6, STEAD, STn, survivin, syk-ZAP70, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TACSTD1 (tumor associated calcium signal transducer 1), TACSTD2, TAG-72-4, TAGE, TARP (T cell receptor gamma alternate reading frame protein), TEL/AML1 fusion protein, TEM1, TEM8 (endosialin or CD248), TGFβ, TIE2, TLP, TMPRSS2 ETS fusion gene, TNF-receptor (TNF-α receptor, TNF-β receptor; or TNF-γ receptor), transferrin receptor, TPS, TRP-1 (tyrosine related protein 1), TRP-2, TRP-2/INT2, TSP-180, VEGF receptor, WNT, WT-1 (Wilm's tumor antigen) and XAGE.

In one aspect, the payload of the present invention may be a TCR specifically recognizing Her2/neu epitope which has nucleic acid sequences of α chain and β chain disclosed in U.S. Patent Publication NO.: US20110280894 and International Patent Publication NO. WO2016133779A1; the contents of each of which are incorporated herein by reference in their entirety. In another aspect, the payload of the present invention may be a TCR specific to TSA tyrosinase (See U.S. Pat. No. 8,697,854; the contents of which are incorporated herein by reference in their entirety).

In other aspects, payloads of the present invention may be TCRs having polypeptide sequences specific to synovial sarcoma X Breakpoint (SSX)-2 antigen (U.S. Pat. No. 9,345,748); human papillomavirus (HPV) 16 E6 antigen (International Patent Publication NO.: WO2015/009606); cytomegalovirus (CMV) phosphoprotein pp65 (U.S. Pat. No. 8,722,048); and WT-1 specific TCR comprising a TCR α-chain having an amino acid sequence as set forth in any one of SEQ ID NOs.: 5-8, and comprising a TCR β-chain having an amino acid sequence as set forth in SEQ ID NO.: 12 or 13, as disclosed in US patent publication NO.: US2016/0083449; the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the TCR specific to a TSA may be modified to possess a sequence encoding an affinity weakening motif which imparts a reduction in non-specific binding to a TSA. In some embodiments, an affinity weakening motif having a modification to a TCR CDR1 or CDR2 region may be used to weaken the interaction between TCR and HLA proteins (see, International Patent Publication NO.: WO2016/014725; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the TCR specific to a TSA may be modified to possess an affinity enhancing motif which imparts an enhancement of binding specificity and affinity for a target antigen. In some embodiments, such high affinity TCRs may be generated by using TCR α-chain to select de novo generated TCR β-chains that pair with an antigen specific TCR α-chain during T cell development in vitro to form enhanced TCRs (see, International Patent Publication NO.: WO2013/166321; the contents of which are incorporated herein by reference in their entirety). In other embodiments, the modified TCR may also possess a sequence encoding an affinity enhancing modification CDR3 region which strengthens the interaction between the TCR and the TSA.

In one embodiment, the TCR specific to a TSA may be modified using zinc finger nucleases (ZFNs), TALENs or a CRISPR/Cas system. The TCR α chain and (3 chain may contain target sites of a nuclease. The nuclease can cleave the TCR sequence causing a certain degree of disruption of the TCR (see US Patent Publication NO.: US2014/0301990; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, the TCR specific to a TSA may be a soluble single-chain TCR having the structure: Vα2-L-Vβ or Vβ-L-Vα2, wherein L is a linker peptide that links a TCR variable β region (Vβ) with a TCR variable α region of the family 2 (Vα2), as discussed in International Patent Publication NO.: WO2011/044186; the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the TCR specific to a TSA may be maturated to increase its affinity to the TSA according to methods described in US Patent Publication NO.: US2014/0065111; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the TCRs may be specific to the Fc domain of an antibody (e.g. FcgR1a) and utilized to enhance efficacy of antibody mediated therapy, as discussed in International Patent Publication NO.: WO2015/179833; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, payloads of the present invention may be recombinant constructs which act as trifunctional T-cell signaling couplers (TriTACs) mimicking the naturally signaling through T cell receptors. The TriTACs enhance chimeric receptor activity while retaining WIC unrestricted targeting. In some aspects, the recombinant construct may comprise a target specific binding ligand such as a scFV specific to a TSA or a designed ankyrin repeat (DARPin), a ligand that binds a protein associated with the TCR complex and a TCR signaling domain polypeptide, e.g. as described in the International Patent Application NO.: WO2015/117229; the contents of which are incorporated herein by reference in their entirety. The TCR associated proteins may be selected from CD3, ZAP70 9zeta-chain associated protein kinase 70), TYN and CD247. In some aspects, the ligand of a TriTAC that binds the TCR associated protein may be an antibody or an antibody fragment (e.g., scFv). In other aspects, the TCR signaling domain polypeptide of a TriTAC may comprise the transmembrane and cytoplasmic domain of CD4.

According to the present invention, the α chain and β chain of the TCR of the present invention may be included in separate constructs, for example as payloads of two effector modules. In other embodiments, the α chain and β chain of the TCR of the present invention may be included in a single effector module as two payloads of the same effector module, for example as illustrated in FIGS. 3-6 .

5. Chimeric Antigen Receptors (CARS)

In some embodiments, payloads of the present invention may be a chimeric antigen receptors (CARs) which when transduced into immune cells (e.g., T cells and NK cells), can re-direct the immune cells against the target (e.g., a tumor cell) which expresses a molecule recognized by the extracellular target moiety of the CAR.

As used herein, the term “chimeric antigen receptor (CAR)” refers to a synthetic receptor that mimics TCR on the surface of T cells. In general, a CAR is composed of an extracellular targeting domain, a transmembrane domain/region and an intracellular signaling/activation domain. In a standard CAR receptor, the components: the extracellular targeting domain, transmembrane domain and intracellular signaling/activation domain, are linearly constructed as a single fusion protein. The extracellular region comprises a targeting domain/moiety (e.g., a scFv) that recognizes a specific tumor antigen or other tumor cell-surface molecules. The intracellular region may contain a signaling domain of TCR complex (e.g., the signal region of CD3ζ), and/or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX-40 (CD134). For example, a “first-generation CAR” only has the CD3ζ signaling domain, whereas in an effort to augment T-cell persistence and proliferation, costimulatory intracellular domains are added, giving rise to second generation CARs having a CD3ζsignal domain plus one costimulatory signaling domain, and third generation CARs having CD3ζ signal domain plus two or more costimulatory signaling domains. A CAR, when expressed by a T cell, endows the T cell with antigen specificity determined by the extracellular targeting moiety of the CAR. Recently, it is also desirable to add one or more elements such as homing and suicide genes to develop a more competent and safer architecture of CAR, so called the fourth-generation CAR.

Cells such as T cells engineered to express a CAR can be redirected to attack target cells that express a molecule which can be recognized by the targeting moiety of the CAR.

In some embodiments, the extracellular targeting domain is joined through the hinge (also called space domain or spacer) and transmembrane regions to an intracellular signaling domain. The hinge connects the extracellular targeting domain to the transmembrane domain which transverses the cell membrane and connects to the intracellular signaling domain. The hinge may need to be varied to optimize the potency of CAR transformed cells toward cancer cells due to the size of the target protein where the targeting moiety binds, and the size and affinity of the targeting domain itself. Upon recognition and binding of the targeting moiety to the target cell, the intracellular signaling domain leads to an activation signal to the CAR T cell, which is further amplified by the “second signal” from one or more intracellular costimulatory domains. The CAR T cell, once activated, can destroy the target cell.

In some embodiments, the CAR of the present invention may be split into two parts, each part is linked a dimerizing domain, such that an input that triggers the dimerization promotes assembly of the intact functional receptor. Wu and Lim recently reported a split CAR in which the extracellular CD19 binding domain and the intracellular signaling element are separated and linked to the FKBP domain and the FRB* (T2089L mutant of FKBP-rapamycin binding) domain that heterodimerize in the presence of the rapamycin analog AP21967. The split receptor is assembled in the presence of AP21967 and together with the specific antigen binding, activates T cells (Wu et al., Science, 2015, 625(6258): aab4077).

In some embodiments, the CAR of the present invention may be designed as an inducible CAR. Sakemura et al recently reported the incorporation of a Tet-On inducible system to the CD19 CAR construct. The CD19 CAR is activated only in the presence of doxycycline (Dox). Sakemura reported that Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD19⁺ cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells (Sakemura et al., Cancer Immuno. Res., 2016, Jun. 21, Epub ahead of print). In one example, this Tet-CAR may be the payload of the effector module under the control of SREs (e.g., DDs) of the invention. The dual systems provide more flexibility to turn-on and off the CAR expression in transduced T cells.

According to the present invention, the payload of the present invention may be a first-generation CAR, or a second-generation CAR, or a third-generation CAR, or a fourth-generation CAR. Representative effector module embodiments comprising CAR constructs are illustrated in FIGS. 13-18 . In some embodiments, the payload of the present invention may be a full CAR construct composed of the extracellular domain, the hinge and transmembrane domain and the intracellular signaling region. In other embodiments, the payload of the present invention may be a component of the full CAR construct including an extracellular targeting moiety, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more co-stimulatory domain, and other additional elements that improve CAR architecture and functionality including but not limited to a leader sequence, a homing element and a safety switch, or the combination of such components.

CARs regulated by biocircuits and compositions of the present invention are tunable and thereby offer several advantages. The reversible on-off switch mechanism allows management of acute toxicity caused by excessive CAR-T cell expansion. Pulsatile CAR expression using SREs of the present invention may be achieved by cycling ligand level. The ligand conferred regulation of the CAR may be effective in offsetting tumor escape induced by antigen loss, avoiding functional exhaustion caused by tonic signaling due to chronic antigen exposure and improving the persistence of CAR expressing cells in vivo.

In some embodiments, biocircuits and compositions of the invention may be utilized to down regulate CAR expression to limit on target on tissue toxicity caused by tumor lysis syndrome. Down regulating the expression of the CARs of the present invention following anti-tumor efficacy may prevent (1) On target off tumor toxicity caused by antigen expression in normal tissue. (2) antigen independent activation in vivo.

Extracellular Targeting Domain/Moiety

In accordance with the invention, the extracellular target moiety of a CAR may be any agent that recognizes and binds to a given target molecule, for example, a neoantigen on tumor cells, with high specificity and affinity. The target moiety may be an antibody and variants thereof that specifically binds to a target molecule on tumor cells, or a peptide aptamer selected from a random sequence pool based on its ability to bind to the target molecule on tumor cells, or a variant or fragment thereof that can bind to the target molecule on tumor cells, or an antigen recognition domain from native T-cell receptor (TCR) (e.g. CD4 extracellular domain to recognize HIV infected cells), or exotic recognition components such as a linked cytokine that leads to recognition of target cells bearing the cytokine receptor, or a natural ligand of a receptor.

In some embodiments, the targeting domain of a CAR may be a Ig NAR, a Fab fragment, a Fab′ fragment, a F(ab)′2 fragment, a F(ab)′3 fragment, Fv, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a unitbody, a nanobody, or an antigen binding region derived from an antibody that specifically recognizes a target molecule, for example a tumor specific antigen (TSA). In one embodiment, the targeting moiety is a scFv antibody. The scFv domain, when it is expressed on the surface of a CAR T cell and subsequently binds to a target protein on a cancer cell, is able to maintain the CAR T cell in proximity to the cancer cell and to trigger the activation of the T cell. A scFv can be generated using routine recombinant DNA technology techniques and is discussed in the present invention.

In one embodiment, the targeting moiety of a CAR construct may be an aptamer such as a peptide aptamer that specifically binds to a target molecule of interest. The peptide aptamer may be selected from a random sequence pool based on its ability to bind to the target molecule of interest.

In some embodiments, the targeting moiety of a CAR construct may be a natural ligand of the target molecule, or a variant and/or fragment thereof capable of binding the target molecule. In some aspects, the targeting moiety of a CAR may be a receptor of the target molecule, for example, a full-length human CD27, as a CD70 receptor, may be fused in frame to the signaling domain of CD3 forming a CD27 chimeric receptor as an immunotherapeutic agent for CD70-positive malignancies (See, e.g., US patent publication NO.: US20130323214; the contents of which are incorporated by reference herein in their entirety).

In some embodiments, the targeting moiety of a CAR may recognize a tumor specific antigen (TSA), for example a cancer neoantigen which is restrictedly expressed on tumor cells.

As non-limiting examples, the CAR of the present invention may comprise the extracellular targeting domain capable of binding to a tumor specific antigen selected from 5T4, 707-AP, A33, AFP (α-fetoprotein), AKAP-4 (A kinase anchor protein 4), ALK, α5β1-integrin, androgen receptor, annexin II, alpha-actinin-4, ART-4, B1, B7H3, B7H4, BAGE (B melanoma antigen), BCMA, BCR-ABL fusion protein, beta-catenin, BKT-antigen, BTAA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calretinin, CAIX (carbonic anhydrase), CAMEL (cytotoxic T-lymphocyte recognized antigen on melanoma), CAM43, CAP-1, Caspase-8/m, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD25, CD27/m, CD28, CD30, CD33, CD34, CD36, CD38, CD40/CD154, CD41, CD44v6, CD44v7/8, CD45, CD49f, CD56, CD68\KP1, CD74, CD79a/CD79b, CD103, CD123, CD133, CD138, CD171, cdc27/m, CDK4 (cyclin dependent kinase 4), CDKN2A, CDS, CEA (carcinoembryonic antigen), CEACAM5, CEACAM6, chromogranin, c-Met, c-Myc, coa-1, CSAp, CT7, CT10, cyclophilin B, cyclin B1, cytoplasmic tyrosine kinases, cytokeratin, DAM-10, DAM-6, dek-can fusion protein, desmin, DEPDC1 (DEP domain containing 1), E2A-PRL, EBNA, EGF-R (epidermal growth factor receptor), EGP-1 (epithelial glycoprotein −1) (TROP-2), EGP-2, EGP-40, EGFR (epidermal growth factor receptor), EGFRvIII, EF-2, ELF2M, EMMPRIN, EpCAM (epithelial cell adhesion molecule), EphA2, Epstein Barr virus antigens, Erb (ErbB 1; ErbB3; ErbB4), ETA (epithelial tumor antigen), ETV6-AML1 fusion protein, FAP (fibroblast activation protein), FBP (folate-binding protein), FGF-5, folate receptor α, FOS related antigen 1, fucosyl GM1, G250, GAGE (GAGE-1; GAGE-2), galactin, GD2 (ganglioside), GD3, GFAP (glial fibrillary acidic protein), GM2 (oncofetal antigen-immunogenic-1; OFA-I-1), GnT-V, Gp100, H4-RET, HAGE (helicase antigen), HER-2/neu, HIFs (hypoxia inducible factors), HIF-1α, HIF-2a, HLA-A2, HLA-A*0201-R170I, HLA-A11, HMWMAA, Hom/Mel-40, HSP70-2M (Heat shock protein 70), HST-2, HTgp-175, hTERT (or hTRT), human papillomavirus-E6/human papillomavirus-E7 and E6, iCE (immune-capture EIA), IGF-1R, IGH-IGK, IL2R, ILS, ILK (integrin-linked kinase), IMP3 (insulin-like growth factor II mRNA-binding protein 3), IRF4 (interferon regulatory factor 4), KDR (kinase insert domain receptor), KIAA0205, KRAB-zinc finger protein (KID)-3; KID31, KSA (17-1A), K-ras, LAGE, LCK, LDLR/FUT (LDLR-fucosyltransferaseAS fusion protein), LeY (Lewis Y), MAD-CT-1, MAGE (tyrosinase, melanoma-associated antigen) (MAGE-1; MAGE-3), melan-A tumor antigen (MART), MART-2/Ski, MC1R (melanocortin 1 receptor), MDM2, mesothelin, MPHOSPH1, MSA (muscle-specific actin), mTOR (mammalian targets of rapamycin), MUC-1, MUC-2, MUM-1 (melanoma associated antigen (mutated) 1), MUM-2, MUM-3, Myosin/m, MYL-RAR, NA88-A, N− acetylglucosaminyltransferase, neo-PAP, NF-KB (nuclear factor-kappa B), neurofilament, NSE (neuron-specific enolase), Notch receptors, NuMa, N-Ras, NY-BR-1, NY-CO-1, NY-ESO-1, Oncostatin M, OS-9, OY-TES1, p53 mutants, p190 minor bcr-ab1, p15(58), p185erbB2, p180erbB-3, PAGE (prostate associated gene), PAP (prostatic acid phosphatase), PAX3, PAX5, PDGFR (platelet derived growth factor receptor), cytochrome P450 involved in piperidine and pyrrolidine utilization (PIPA), Pm1-RAR alpha fusion protein, PR-3 (proteinase 3), PSA (prostate specific antigen), PSM, PSMA (Prostate stem cell antigen), PRAME (preferentially expressed antigen of melanoma), PTPRK, RAGE (renal tumor antigen), Raf (A-Raf, B-Raf and C-Raf), Ras, receptor tyrosine kinases, RCAS1, RGSS, ROR1 (receptor tyrosine kinase-like orphan receptor 1), RU1, RU2, SAGE, SART-1, SART-3, SCP-1, SDCCAG16, SP-17 (sperm protein 17), src-family, SSX (synovial sarcoma X breakpoint)-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, STAT-3, STAT-5, STAT-6, STEAD, STn, survivin, syk-ZAP70, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TACSTD1 (tumor associated calcium signal transducer 1), TACSTD2, TAG-72-4, TAGE, TARP (T cell receptor gamma alternate reading frame protein), TEL/AML1 fusion protein, TEM1, TEM8 (endosialin or CD248), TGFβ, TIE2, TLP, TMPRSS2 ETS fusion gene, TNF-receptor (TNF-α receptor, TNF-β receptor; or TNF-γ receptor), transferrin receptor, TPS, TRP-1 (tyrosine related protein 1), TRP-2, TRP-2/INT2, TSP-180, VEGF receptor, WNT, WT-1 (Wilm's tumor antigen) and XAGE.

In some embodiments, the CAR of the present invention may comprise a universal immune receptor which has a targeting moiety capable of binding to a labelled antigen. Methods of generating universal immune receptor CAR are discussed in International Patent Publication NO.: WO2013044225A1; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the CAR of the present invention may comprise the targeting moiety capable of binding to a pathogen antigen.

In some embodiments, the CAR of the present invention may comprise the targeting moiety capable of binding to non-protein molecules such as tumor-associated glycolipids and carbohydrates. TSAs may also be lipid molecules, polysaccharides, saccharides, nucleic acids, haptens, carbohydrate, or the combinations thereof.

In some embodiments, the CAR of the present invention may comprise the targeting moiety capable of binding to a component within the tumor microenvironment including proteins expressed in various tumor stroma cells including tumor associated macrophages (TAMs), immature monocytes, immature dendritic cells, immunosuppressive CD4⁺CD25⁺ regulatory T cells (Treg) and MDSCs. A recent study using an animal model, demonstrated that after systemic transplantation, T cells expressing VEGFR-2 CAR and IL12 infiltrated the tumors, expanded and persisted within tumor mass leading to tumor regression. The anti-tumor effect was dependent on targeting of IL12-responsive host cells via activation of VEGFR-2 CAR-T cells and release of IL12 (Chinnasamy et al., Clinical Cancer Research, 2012, 18: 1672-1683).

In some embodiments, the CAR of the present invention may comprise the targeting moiety capable of binding to a cell surface adhesion molecule, a surface molecule of an inflammatory cell that appears in an autoimmune disease, or a TCR causing autoimmunity.

As non-limiting examples, the targeting moiety of the present invention may be a scFv antibody that recognizes a tumor specific antigen (TSA), for example scFvs of antibodies SS, SS1 and HN1 that specifically recognize and bind to human mesothelin (U.S. Pat. No. 9,359,447), scFv of antibody of GD2 (U.S. Pat. No. 9,315,585), a CD19 antigen binding domain (U.S. Pat. NO.: 9, 328, 156); a NKG2D ligand binding domain (U.S. Pat. No. 9,273,283; US patent publication NO.: US20160311906A1); human anti-mesothelin scFvs comprising the amino acid sequences of SEQ ID Nos.: 11 and 12 of U.S. Pat. No. 9,272,002; an anti-CS1 binding agent (US patent publication NO.: US20160075784); an anti-BCMA binding domain (International Patent Publication NO.: WO2016/014565); anti-CD19 scFv antibody of SEQ ID NO.: 20 in U.S. Pat. No. 9,102,761; GFR alpha 4 antigen binding fragments having the amino acid sequences of SEQ ID NOs: 59 and 79 of International patent publication NO.: 2016/025880; anti-CLL-1 (C-type lectin-like molecule 1) binding domains having the amino acid sequences of SEQ ID NO:47, 44, 48, 49, 50, 39, 40, 41, 42, 43, 45, 46, 51, 73, 70, 74, 75, 76, 65, 66, 67, 68, 69, 71, 72, 77, 195, 86, 83, 87, 88, 89, 78, 79, 80, 81, 82, 84, 85, 90 and 196 of International Patent Publication NO.: WO2016014535); CD33 binding domains having the amino acid sequences of SEQ ID NOs: 39-46 of International patent publication NO.: WO2016014576; a GPC3 (glypican-3) binding domain (SEQ ID NO.: 2 and SEQ ID NO.: 4 of International patent publication NO.: WO2016036973); a GFR alpha4 (Glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 cell-surface receptor) binding domain (International Patent Publication NO.: WO2016025880); CD123 binding domains having the amino acid sequences of SEQ ID NOs: 480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, and 275 of International patent publication NO.: WO20160258896; an anti-ROR1 antibody or fragments thereof (International patent publication NO.: WO2016016344); scFvs specific to GPC-3 (SEQ ID NOs: 1 and 24 of International patent publication NO.: WO2016049459); scFv for CSPG4 (SEQ ID NO.: 2 of International patent publication NO.: WO2015080981; scFv for folate receptor alpha (US Patent Publication NO.: US20170002072A1); the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, natural ligands may be used as the targeting moieties of the CARs of the present invention. Such natural ligands may be capable of binding to the antigens with affinity in the range of the scFvs and can redirect T cells specificity and effector functions to target cells expressing the complementary receptor. In some embodiments, the targeting moiety of the CAR may be neuregulin-1 (NRG1) which is a natural ligand for HER3 and HER4; VEGF which is a natural ligand of VEGFR; IL13 wildtype protein or IL13 mutein e.g E13Y which binds to IL13Ra2; NKG2D ligand, which is a natural ligand of NKG2D receptor; CD70 which is ligand of CD27; and a proliferation-inducing ligand (APRIL) which is a natural high affinity ligand for BCMA8 and transmembrane activator and CAML interactor (TACI). Any of the ligand based BCMA CARs taught in the US Patent Publication No. US20160362467A1, the contents of which are incorporated by reference in their entirety.

In some embodiments, the targeting moieties of the present invention may be scFv comprising the amino acid sequences in Table 11.

TABLE 11 scFv sequences Target Description SEQ ID NO Source Activated alpha.v. Beta.3 scFv 5474 SEQ ID NO. 8 in US20090117096A1 integrin receptor Activated alpha.v. Beta.3 scFv 5475 SEQ ID NO. 2 in US20090117096A1 integrin receptor Activated alpha.v. Beta.3 scFv 5476 SEQ ID NO. 4 in US20090117096A1 integrin receptor Adalimumab scFv 5477 SEQ ID NO. 41 in US20160208021 Adalinumab scFv 5478 SEQ ID NO. 41 in WO2016112870 ALK scFv 5479 SEQ ID NO. 17 in WO2015069922 ALK scFv 5480 SEQ ID NO. 18 in WO2015069922 ALK scFv 5481 SEQ ID NO. 19 in WO2015069922 ALK scFv 5482 SEQ ID NO. 20 in WO2015069922 ALK scFv 5483 SEQ ID NO. 21 in WO2015069922 ALK scFv 5484 SEQ ID NO. 22 in WO2015069922 ALK scFv 5485 SEQ ID NO. 23 in WO2015069922 ALK scFv 5486 SEQ ID NO. 17 in US20160280798A1 ALK scFv 5487 SEQ ID NO. 18 in US20160280798A1 ALK scFv 5488 SEQ ID NO. 19 in US20160280798A1 ALK scFv 5489 SEQ ID NO. 20 in US20160280798A1 ALK scFv 5490 SEQ ID NO. 21 in US20160280798A1 ALK scFv 5491 SEQ ID NO. 22 in US20160280798A1 ALK scFv 5492 SEQ ID NO. 23 in US20160280798A1 ALK scFv 5493 SEQ ID NO. 24 in US20160280798A1 ALK scFv 5494 SEQ ID NO. 24 in WO2015069922 B7H3 scFv 5495 SEQ ID NO. 100 in WO2016033225 B7H3 scFv 5496 SEQ ID NO. 101 in WO2016033225 B7H3 scFv 5497 SEQ ID NO. 102 in WO2016033225 B7H3 scFv 5498 SEQ ID NO. 103 in WO2016033225 B7H3 scFv 5499 SEQ ID NO. 104 in WO2016033225 B7H3 scFv 5500 SEQ ID NO. 105 in WO2016033225 B7H3 scFv 5501 SEQ ID NO. 17 in WO2016033225 B7H3 scFv 5502 SEQ ID NO. 18 in WO2016033225 B7H3 scFv 5503 SEQ ID NO. 19 in WO2016033225 B7H3 scFv 5504 SEQ ID NO. 20 in WO2016033225 B7H3 scFv 5505 SEQ ID NO. 21 in WO2016033225 B7H3 scFv 5506 SEQ ID NO. 22 in WO2016033225 B7H3 scFv 5507 SEQ ID NO. 23 in WO2016033225 B7H3 scFv 5508 SEQ ID NO. 24 in WO2016033225 B7H3 scFv 5509 SEQ ID NO. 25 in WO2016033225 B7H3 scFv 5510 SEQ ID NO. 26 in WO2016033225 B7H3 scFv 5511 SEQ ID NO. 27 in WO2016033225 B7H3 scFv 5512 SEQ ID NO. 87 in WO2016033225 B7H3 scFv 5513 SEQ ID NO. 88 in WO2016033225 B7H3 scFv 5514 SEQ ID NO. 89 in WO2016033225 B7H3 scFv 5515 SEQ ID NO. 90 in WO2016033225 B7H3 scFv 5516 SEQ ID NO. 91 in WO2016033225 B7H3 scFv 5517 SEQ ID NO. 92 in WO2016033225 B7H3 scFv 5518 SEQ ID NO. 94 in WO2016033225 B7H3 scFv 5519 SEQ ID NO. 95 in WO2016033225 B7H3 scFv 5520 SEQ ID NO. 96 in WO2016033225 B7H3 scFv 5521 SEQ ID NO. 97 in WO2016033225 B7H3 scFv 5522 SEO ID NO. 98 in WO2016033225 B7H3 scFv 5523 SEQ ID NO. 99 in WO2016033225 B7H4 scFv 5524 SEQ ID NO. 1 in WO2013067492 B7H4 scFv 5525 SEQ ID NO. 2 in WO2013067492 B7H4 scFv 5526 SEQ ID NO. 3 in WO2013067492 B7H4 scFv 5527 SEQ ID NO. 4 in WO2013067492 B7H4 scFv 5528 SEQ ID NO. 1 in U.S. Pat. No. 9,422,351B2 BCMA scFv 5529 SEQ ID NO. 152 in WO2016168595A1 BCMA scFv 5530 SEQ ID NO. 158 in WO2016168595A1 BCMA scFv 5531 SEQ ID NO. 176 in WO2016168595A1 BCMA scFv 5532 SEQ ID NO. 182 in WO2016168595A1 BCMA scFv 5533 SEQ ID NO. 188 in WO2016168595A1 BCMA scFv 5534 SEQ ID NO. 200 in WO2016168595A1 BCMA scFv 5535 SEQ ID NO. 212 in WO2016168595A1 BCMA scFv 5536 SEQ ID NO. 218 in WO2016168595A1 BCMA scFv 5537 SEQ ID NO. 224 in WO2016168595A1 BCMA scFv 5538 SEQ ID NO. 284 in WO2016168595A1 BCMA scFv 5539 SEQ ID NO. 290 in WO2016168595A1 BCMA scFv 5540 SEQ ID NO. 296 in WO2016168595A1 BCMA scFv 5541 SEQ ID NO. 302 in WO2016168595A1 BCMA scFv 5542 SEQ ID NO. 314 in WO2016168595A1 BCMA scFv 5543 SEQ ID NO. 326 in WO2016168595A1 BCMA scFv 5544 SEQ ID NO. 344 in WO2016168595A1 BCMA scFv 5545 SEQ ID NO. 129 in WO2016014565 BCMA scFv 5546 SEQ ID NO. 130 in WO2016014565 BCMA scFv 5547 SEQ ID NO. 131 in WO2016014565 BCMA scFv 5548 SEQ ID NO. 132 in WO2016014565 BCMA scFv 5549 SEQ ID NO. 133 in WO2016014565 BCMA scFv 5550 SEQ ID NO. 134 in WO2016014565 BCMA scFv 5551 SEQ ID NO. 135 in WO2016014565 BCMA scFv 5552 SEQ ID NO. 136 in WO2016014565 BCMA scFv 5553 SEQ ID NO. 138 in WO2016014565 BCMA scFv 5554 SEQ ID NO. 139 in WO2016014565 BCMA scFv 5555 SEQ ID NO. 140 in WO2016014565 BCMA scFv 5556 SEQ ID NO. 141 in WO2016014565 BCMA scFv 5557 SEQ ID NO. 142 in WO2016014565 BCMA scFv 5558 SEQ ID NO. 143 in WO2016014565 BCMA scFv 5559 SEQ ID NO. 144 in WO2016014565 BCMA scFv 5560 SEQ ID NO. 145 in WO2016014565 BCMA scFv 5561 SEQ ID NO. 146 in WO2016014565 BCMA scFv 5562 SEQ ID NO. 147 in WO2016014565 BCMA scFv 5563 SEQ ID NO. 148 in WO2016014565 BCMA scFv 5564 SEQ ID NO. 149 in WO2016014565 BCMA scFv 5565 SEQ ID NO. 263 in WO2016014565 BCMA scFv 5566 SEQ ID NO. 264 in WO2016014565 BCMA scFv 5567 SEQ ID NO. 265 in WO2016014565 BCMA scFv 5568 SEQ ID NO. 266 in WO2016014565 BCMA scFv 5569 SEQ ID NO. 271 in WO2016014565 BCMA scFv 5570 SEQ ID NO. 273 in WO2016014565 BCMA scFv 5571 SEQ ID NO. 273 in WO2016014565 BCMA scFv 5572 SEQ ID NO. 39 in WO2016014565 BCMA scFv 5573 SEQ ID NO. 40 in WO2016014565 BCMA scFv 5574 SEQ ID NO. 41 in WO2016014565 BCMA scFv 5575 SEQ ID NO. 42 in WO2016014565 BCMA scFv 5576 SEQ ID NO. 43 in WO2016014565 BCMA scFv 5577 SEQ ID NO. 44 in WO2016014565 BCMA scFv 5578 SEQ ID NO. 45 in WO2016014565 BCMA scFv 5579 SEQ ID NO. 46 in WO2016014565 BCMA scFv 5580 SEQ ID NO. 47 in WO2016014565 BCMA scFv 5581 SEQ ID NO. 48 in WO2016014565 BCMA scFv 5582 SEQ ID NO. 49 in WO2016014565 BCMA scFv 5583 SEQ ID NO. 50 in WO2016014565 BCMA scFv 5584 SEQ ID NO. 51 in WO2016014565 BCMA scFv 5585 SEQ ID NO. 52 in WO2016014565 BCMA scFv 5586 SEQ ID NO. 53 in WO2016014565 BCMA scFv 5587 SEQ ID NO. 64 in WO2016014565 BCMA scFv 5588 SEQ ID NO. 129 in WO2016014565 BCMA scFv 5589 SEQ ID NO. 130 in WO2016014565 BCMA scFv 5590 SEQ ID NO. 131 in WO2016014565 BCMA scFv 5591 SEQ ID NO. 132 in WO2016014565 BCMA scFv 5592 SEQ ID NO. 133 in WO2016014565 BCMA scFv 5593 SEQ ID NO. 134 in WO2016014565 BCMA scFv 5594 SEQ ID NO. 135 in WO2016014565 BCMA scFv 5595 SEQ ID NO. 136 in WO2016014565 BCMA scFv 5596 SEQ ID NO. 137 in WO2016014565 BCMA scFv 5597 SEQ ID NO. 138 in WO2016014565 BCMA scFv 5598 SEQ ID NO. 139 in WO2016014565 BCMA scFv 5599 SEQ ID NO. 140 in WO2016014565 BCMA scFv 5600 SEQ ID NO. 141 in WO2016014565 BCMA scFv 5601 SEQ ID NO. 142 in WO2016014565 BCMA scFv 5602 SEQ ID NO. 143 in WO2016014565 BCMA scFv 5603 SEQ ID NO. 144 in WO2016014565 BCMA scFv 5604 SEQ ID NO. 145 in WO2016014565 BCMA scFv 5605 SEQ ID NO. 146 in WO2016014565 BCMA scFv 5606 SEQ ID NO. 147 in WO2016014565 BCMA scFv 5607 SEQ ID NO. 148 in WO2016014565 BCMA scFv 5608 SEQ ID NO. 149 in WO2016014565 BCMA scFv 5609 SEQ ID NO. 263 in WO2016014565 BCMA scFv 5610 SEQ ID NO. 264 in WO2016014565 BCMA scFv 5611 SEQ ID NO. 265 in WO2016014565 BCMA scFv 5612 SEQ ID NO. 266 in WO2016014565 BCMA scFv 5613 SEQ ID NO. 39 in WO2016014565 BCMA scFv 5614 SEQ ID NO. 40 in WO2016014565 BCMA scFv 5615 SEQ ID NO. 41 in WO2016014565 BCMA scFv 5616 SEQ ID NO. 42 in WO2016014565 BCMA scFv 5617 SEQ ID NO. 43 in WO2016014565 BCMA scFv 5618 SEQ ID NO. 44 in WO2016014565 BCMA scFv 5619 SEQ ID NO. 45 in WO2016014565 BCMA scFv 5620 SEQ ID NO. 46 in WO2016014565 BCMA scFv 5621 SEQ ID NO. 47 in WO2016014565 BCMA scFv 5622 SEQ ID NO. 48 in WO2016014565 BCMA scFv 5623 SEQ ID NO. 49 in WO2016014565 BCMA scFv 5624 SEQ ID NO. 50 in WO2016014565 BCMA scFv 5625 SEQ ID NO. 51 in WO2016014565 BCMA scFv 5626 SEQ ID NO. 52 in WO2016014565 BCMA scFv 5627 SEQ ID NO. 53 in WO2016014565 BCMA scFv 5628 SEQ ID NO. 214 in US20160311907A1 BCMA scFv 5629 SEQ ID NO. 215 in US20160311907A1 BCMA scFv 5630 SEQ ID NO. 216 in US20160311907A1 BCMA scFv 5631 SEQ ID NO. 217 in US20160311907A1 BCMA scFv 5632 SEQ ID NO. 218 in US20160311907A1 BCMA scFv 5633 SEQ ID NO. 219 in US20160311907A1 BCMA scFv 5634 SEQ ID NO. 220 in US20160311907A1 BCMA scFv 5635 SEQ ID NO. 221 in US20160311907A1 BCMA scFv 5636 SEQ ID NO. 222 in 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5959 SEQ ID NO. 162 in WO2016115482A1 CD33 scFv 5960 SEQ ID NO. 163 in WO2016115482A1 CD33/CD3sBiSpecifc scFv 5961 SEQ ID NO. 33 in WO2014144722A2 CD33/CD3sBiSpecifc scFv 5962 SEQ ID NO. 34 in WO2014144722A2 CD33/CD3sBiSpecifc scFv 5963 SEQ ID NO. 84 in WO2014144722A2 CD37 5964 SEQ ID NO. 21 in US20170000900 CD37 5965 SEQ ID NO. 22 in US20170000900 CD44 scFv 5966 SEQ ID NO. 17 in WO2016042461A1 CD46 scFv 5967 SEQ ID NO. in WO2016040683 CD46 scFv 5968 SEQ ID NO. in WO2016040683 CD46 scFv 5969 SEQ ID NO. in WO2016040683 CD46 scFv 5970 SEQ ID NO. in WO2016040683 CD46 scFv 5971 SEQ ID NO. in WO2016040683 CD46 scFv 5972 SEQ ID NO. in WO2016040683 CD46 scFv 5973 SEQ ID NO. in WO2016040683 CD46 scFv 5974 SEQ ID NO. in WO2016040683 CD46 scFv 5975 SEQ ID NO. in WO2016040683 CD46 scFv 5976 SEQ ID NO. in WO2016040683 CD46 scFv 5977 SEQ ID NO. in WO2016040683 CD46 scFv 5978 SEQ ID NO. in WO2016040683 CD46 scFv 5979 SEQ ID NO. in WO2016040683 CD46 scFv 5980 SEQ ID NO. in WO2016040683 CD46 scFv 5981 SEQ ID NO. in WO2016040683 CD46 scFv 5982 SEQ ID NO. in WO2016040683 CD46 scFv 5983 SEQ ID NO. in WO2016040683 CD46 scFv 5984 SEQ ID NO. in WO2016040683 CD46 scFv 5985 SEQ ID NO. in WO2016040683 CD46 scFv 5986 SEQ ID NO. in WO2016040683 CD46 scFv 5987 SEQ ID NO. in WO2016040683 CD46 scFv 5988 SEQ ID NO. in WO2016040683 CD46 scFv 5989 SEQ ID NO. in WO2016040683 CD5 scFv 5990 SEQ ID NO. 16 in WO2016138491 CD79b scFv 5991 SEQ ID NO. 33 in US20160208021 CEA scFv 5992 SEQ ID NO. 1 in US20160303166A1 CEA scFv 5993 SEQ ID NO. 22 in US20140242701A1 CEA scFv 5994 SEQ ID NO. 22 in US20140242701A1 Centuxiamb scFv 5995 SEQ ID NO. 37 in WO2016112870 Centuximab scFv 5996 SEQ ID NO. 37 in US20160208021 Claudin scFv 5997 SEQ ID NO. 11 in WO2016073649A1 Claudin scFv 5998 SEQ ID NO. 17 in WO2014179759A1 Claudin scFv 5999 SEQ ID NO. 5 in WO2016073649A1 Claudin scFv 6000 SEQ ID NO. 7 in WO2016073649A1 Claudin scFv 6001 SEQ ID NO. 9 in WO2016073649A1 Claudin6 scFv 6002 SEQ ID NO. 164 in WO2016115482A1 Claudin7 scFv 6003 SEQ ID NO. 165 in WO2016115482A1 Claudin8 scFv 6004 SEQ ID NO. 166 in WO2016115482A1 CLDN6 scFv 6005 SEQ ID NO. 2 in WO2016150400 CLDN7 scFv 6006 SEQ ID NO. 4 in WO2016150400 CLDN8 scFv 6007 SEQ ID NO. 6 in WO2016150400 CLL1 scFv 6008 SEQ ID NO. 39 in WO2016014535 CLL1 scFv 6009 SEQ ID NO. 40 in WO2016014535 CLL1 scFv 6010 SEQ ID NO. 41 in WO2016014535 CLL1 scFv 6011 SEQ ID NO. 42 in WO2016014535 CLL1 scFv 6012 SEQ ID NO. 43 in WO2016014535 CLL1 scFv 6013 SEQ ID NO. 44 in WO2016014535 CLL1 scFv 6014 SEQ ID NO. 45 in WO2016014535 CLL1 scFv 6015 SEQ ID NO. 46 in WO2016014535 CLL1 scFv 6016 SEQ ID NO. 47 in WO2016014535 CLL1 scFv 6017 SEQ ID NO. 48 in WO2016014535 CLL1 scFv 6018 SEQ ID NO. 49 in WO2016014535 CLL1 scFv 6019 SEQ ID NO. 50 in WO2016014535 CLL1 scFv 6020 SEQ ID NO. 51 in WO2016014535 CLL1 scFv 6021 SEQ ID NO. 200 in US20160311907A1 CLL1 scFv 6022 SEQ ID NO. 201 in US20160311907A1 CLL1 scFv 6023 SEQ ID NO. 202 in US20160311907A1 CLL1 scFv 6024 SEQ ID NO. 203 in US20160311907A1 CLL1 scFv 6025 SEQ ID NO. 204 in US20160311907A1 CLL1 scFv 6026 SEQ ID NO. 205 in US20160311907A1 CLL1 scFv 6027 SEQ ID NO. 206 in US20160311907A1 CLL1 scFv 6028 SEQ ID NO. 207 in US20160311907A1 CLL1 scFv 6029 SEQ ID NO. 208 in US20160311907A1 CLL1 scFv 6030 SEQ ID NO. 209 in US20160311907A1 CLL1 scFv 6031 SEQ ID NO. 210 in US20160311907A1 CLL1 scFv 6032 SEQ ID NO. 211 in US20160311907A1 CLL1 scFv 6033 SEQ ID NO. 212 in US20160311907A1 CLL1 scFv 6034 SEQ ID NO. 213 in US20160311907A1 CMet scFv 6035 SEQ ID NO. 11 in US20040166544 CMet scFv 6036 SEQ ID NO. 12 in US20040166544 CMet scFv 6037 SEQ ID NO. 13 in US20040166544 CMet scFv 6038 SEQ ID NO. 14 in US20040166544 CMet scFv 6039 SEQ ID NO. 15 in US20040166544 CMet scFv 6040 SEQ ID NO. 16 in US20040166544 CMet scFv 6041 SEQ ID NO. 17 in US20040166544 CMet scFv 6042 SEQ ID NO. 18 in US20040166544 CMet scFv 6043 SEQ ID NO. 19 in US20040166544 CMet scFv 6044 SEQ ID NO. 2 in US20040166544 CMet scFv 6045 SEQ ID NO. 21 in US20040166544 CMet scFv 6046 SEQ ID NO. 22 in US20040166544 CMet scFv 6047 SEQ ID NO. 23 in US20040166544 CMet scFv 6048 SEQ ID NO. 25 in US20040166544 CMet scFv 6049 SEQ ID NO. 26 in US20040166544 CMet scFv 6050 SEQ ID NO. 26 in US20150299326 CMet scFv 6051 SEQ ID NO. 27 in US20040166544 CMet scFv 6052 SEQ ID NO. 27 in US20150299326 CMet scFv 6053 SEQ ID NO. 28 in US20040166544 CMet scFv 6054 SEQ ID NO. 28 in US20150299326 CMet scFv 6055 SEQ ID NO. 29 in US20150299326 CMet scFv 6056 SEQ ID NO. 3 in US20040166544 CMet scFv 6057 SEQ ID NO. 30 in US20150299326 CMet scFv 6058 SEQ ID NO. 30 in US20040166544 CMet scFv 6059 SEQ ID NO. 31 in US20040166544 CMet scFv 6060 SEQ ID NO. 32 in US20130034559 CMet scFv 6061 SEQ ID NO. 32 in US20150299326 CMet scFv 6062 SEQ ID NO. 33 in US20040166544 CMet scFv 6063 SEQ ID NO. 34 in US20040166544 CMet scFv 6064 SEQ ID NO. 35 in US20040166544 CMet scFv 6065 SEQ ID NO. 36 in US20040166544 CMet scFv 6066 SEQ ID NO. 37 in US20040166544 CMet scFv 6067 SEQ ID NO. 38 in US20040166544 CMet scFv 6068 SEQ ID NO. 39 in US20040166544 CMet scFv 6069 SEQ ID NO. 4 in US20040166544 CMet scFv 6070 SEQ ID NO. 40 in US20040166544 CMet scFv 6071 SEQ ID NO. 41 in US20040166544 CMet scFv 6072 SEQ ID NO. 42 in US20040166544 CMet scFv 6073 SEQ ID NO. 43 in US20040166544 CMet scFv 6074 SEQ ID NO. 44 in US20040166544 CMet scFv 6075 SEQ ID NO. 48 in US20040166544 CMet scFv 6076 SEQ ID NO. 49 in US20040166544 CMet scFv 6077 SEQ ID NO. 5 in US20040166544 CMet scFv 6078 SEQ ID NO. 50 in US20040166544 CMet scFv 6079 SEQ ID NO. 51 in US20040166544 CMet scFv 6080 SEQ ID NO. 52 in US20040166544 CMet scFv 6081 SEQ ID NO. 53 in US20040166544 CMet scFv 6082 SEQ ID NO. 54 in US20040166544 CMet scFv 6083 SEQ ID NO. 55 in US20040166544 CMet scFv 6084 SEQ ID NO. 56 in US20040166544 CMet scFv 6085 SEQ ID NO. 57 in US20040166544 CMet scFv 6086 SEQ ID NO. 58 in US20040166544 CMet scFv 6087 SEQ ID NO. 6 in US20040166544 CMet scFv 6088 SEQ ID NO. 60 in US20040166544 CMet scFv 6089 SEQ ID NO. 7 in US20040166544 CMet scFv 6090 SEQ ID NO. 9 in US20040166544 CMet scFv 6091 SEQ ID NO. 29 in US20040166544 CS1 scFv 6092 SEQ ID NO. 1 of WO2016090369 CS1 scFv 6093 SEQ ID NO. 17 in WO2014179759A1 CSPG4 scFv 6094 SEQ ID NO. 2 in WO2015080981 CSPG4 scFv 6095 SEQ ID NO. 2 in EP3074025A1 CXCR4 scFv 6096 SEQ ID NO. 83 in US20110020218 CXCR4 scFv 6097 SEQ ID NO. 85 in US20110020218 CXCR4 scFv 6098 SEQ ID NO. 86 in US20110020218 CXCR4 scFv 6099 SEQ ID NO. 89 in US20110020218 E7MC scFv 6100 SEQ ID NO. 223 in WO2016182957A1 E7MC scFv 6101 SEQ ID NO. 224 in WO2016182957A1 E7MC scFv 6102 SEQ ID NO. 225 in WO2016182957A1 E7MC scFv 6103 SEQ ID NO. 226 in WO2016182957A1 E7MC scFv 6104 SEQ ID NO. 227 in WO2016182957A1 E7MC scFv 6105 SEQ ID NO. 228 in WO2016182957A1 E7MC scFv 6106 SEQ ID NO. 229 in WO2016182957A1 E7MC scFv 6107 SEQ ID NO. 230 in WO2016182957A1 E7MC scFv 6108 SEQ ID NO. 231 in WO2016182957A1 E7MC scFv 6109 SEQ ID NO. 232 in WO2016182957A1 EGFR scFv 6110 SEQ ID NO. 11 in WO2014130657 EGFR scFv 6111 SEQ ID NO. 38 in WO2014130657 EGFR scFv 6112 SEQ ID NO. 41 in WO2014130657 EGFR scFv 6113 SEQ ID NO. 44 in WO2014130657 EGFR scFv 6114 SEQ ID NO. 47 in WO2014130657 EGFR scFv 6115 SEQ ID NO. 50 in WO2014130657 EGFR scFv 6116 SEQ ID NO. 53 in WO2014130657 EGFR scFv 6117 SEQ ID NO. 56 in WO2014130657 EGFR scFv 6118 SEQ ID NO. 59 in WO2014130657 EGFR scFv 6119 SEQ ID NO. 62 in WO2014130657 EGFR scFv 6120 SEQ ID NO. 65 in WO2014130657 EGFR scFv 6121 SEQ ID NO. 68 in WO2014130657 EGFR scFv 6122 SEQ ID NO. 71 in WO2014130657 EGFR scFv 6123 SEQ ID NO. 74 in WO2014130657 EGFR scFv 6124 SEQ ID NO. 77 in WO2014130657 EGFR scFv 6125 SEQ ID NO. 80 in WO2014130657 EGFR scFv 6126 SEQ ID NO. 83 in WO2014130657 EGFR scFv 6127 SEQ ID NO. 88 in WO2014130657 EGFR scFv 6128 SEQ ID NO. 91 in WO2014130657 EGFR scFv 6129 SEQ ID NO. 94 in WO2014130657 EGFR scFV 6130 EGFR scFv 6131 EGFR scFv 6132 EGFR scFv 6133 EGFR scFv 6134 EGFR scFv 6135 EGFR scFv 6136 EGFR scFv 6137 EGFR scFv 6138 EGFR scFv 6139 EGFR scFv 6140 EGFR scFv 6141 EGFR scFv 6142 EGFR scFv 6143 EGFR scFv 6144 EGFR scFv 6145 EGFR scFv 6146 EGFR scFv 6147 EGFR scFv 6148 EGFR scFv 6149 EGFR scFv 6150 EGFR scFv 6151 EGFR scFv 6152 EGFR scFv 6153 EGFR scFv 6154 EGFR scFv 6155 EGFR scFv 6156 EGFRvIII scFv 6157 SEQ ID NO. 5 in US20140037628 EGFRvIII scFv 6158 SEQ ID NO. 174 in US20160311907A1 EGFRvIII scFv 6159 SEQ ID NO. 38 in U.S. Pat. No. 9,394,368B2 EGFRvIII scFv 6160 SEQ ID NO. 5 in US20160200819A1 END0180 scFv 6161 SEQ ID NO. 6 in WO2013098813 ERBB2 scFv 6162 SEQ ID NO. 26 in US20110059076A1 ERBB2 scFv 6163 SEQ ID NO. 27 in US20110059076A1 ERBB2 scFv 6164 SEQ ID NO. 1 in U.S. Pat. No. 7,244,826 ERBB2 scFv 6165 SEQ ID NO. 2 in U.S. Pat. No. 7,244,826 ESK/WT scFv 6166 SEQ ID NO. 173 in WO2016115482A1 FcRL5(FcReceptorLike5) scFv 6167 SEQ ID NO. 11 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6168 SEQ ID NO. 15 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6169 SEQ ID NO. 19 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6170 SEQ ID NO. 23 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6171 SEQ ID NO. 27 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6172 SEQ ID NO. 31 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6173 SEQ ID NO. 35 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6174 SEQ ID NO. 39 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6175 SEQ ID NO. 3 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6176 SEQ ID NO. 43 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6177 SEQ ID NO. 7 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6178 SEQ ID NO. 594 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6179 SEQ ID NO. 596 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6180 SEQ ID NO. 598 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6181 SEQ ID NO. 600 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6182 SEQ ID NO. 602 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6183 SEQ ID NO. 604 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6184 SEQ ID NO. 606 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6185 SEQ ID NO. 608 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6186 SEQ ID NO. 610 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6187 SEQ ID NO. 612 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6188 SEQ ID NO. 614 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6189 SEQ ID NO. 616 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6190 SEQ ID NO. 618 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6191 SEQ ID NO. 620 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6192 SEQ ID NO. 622 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6193 SEQ ID NO. 624 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6194 SEQ ID NO. 626 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6195 SEQ ID NO. 628 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6196 SEQ ID NO. 630 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6197 SEQ ID NO. 632 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6198 SEQ ID NO. 634 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6199 SEQ ID NO. 636 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6200 SEQ ID NO. 638 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6201 SEQ ID NO. 640 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6202 SEQ ID NO. 642 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6203 SEQ ID NO. 644 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6204 SEQ ID NO. 646 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6205 SEQ ID NO. 648 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6206 SEQ ID NO. 652 in WO2016090337 FcRL5(FcReceptoiLike5) scFv 6207 SEQ ID NO. 654 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6208 SEQ ID NO. 656 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6209 SEQ ID NO. 658 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6210 SEQ ID NO. 660 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6211 SEQ ID NO. 662 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6212 SEQ ID NO. 664 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6213 SEQ ID NO. 666 in WO2016090337 FcRL5(FcReceptoiLike5) scFv 6214 SEQ ID NO. 668 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6215 SEQ ID NO. 670 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6216 SEQ ID NO. 672 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6217 SEQ ID NO. 674 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6218 SEQ ID NO. 676 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6219 SEQ ID NO. 678 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6220 SEQ ID NO. 680 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6221 SEQ ID NO. 682 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6222 SEQ ID NO. 684 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6223 SEQ ID NO. 686 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6224 SEQ ID NO. 688 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6225 SEQ ID NO. 690 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6226 SEQ ID NO. 692 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6227 SEQ ID NO. 694 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6228 SEQ ID NO. 696 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6229 SEQ ID NO. 700 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6230 SEQ ID NO. 702 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6231 SEQ ID NO. 704 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6232 SEQ ID NO. 706 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6233 SEQ ID NO. 708 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6234 SEQ ID NO. 710 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6235 SEQ ID NO. 712 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6236 SEQ ID NO. 714 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6237 SEQ ID NO. 716 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6238 SEQ ID NO. 718 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6239 SEQ ID NO. 720 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6240 SEQ ID NO. 722 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6241 SEQ ID NO. 724 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6242 SEQ ID NO. 726 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6243 SEQ ID NO. 728 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6244 SEQ ID NO. 730 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6245 SEQ ID NO. 732 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6246 SEQ ID NO. 734 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6247 SEQ ID NO. 736 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6248 SEQ ID NO. 738 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6249 SEQ ID NO. 740 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6250 SEQ ID NO. 742 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6251 SEQ ID NO. 744 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6252 SEQ ID NO. 746 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6253 SEQ ID NO. 748 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6254 SEQ ID NO. 750 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6255 SEQ ID NO. 752 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6256 SEQ ID NO. 754 in WO2016090337 FcRL5(FcReceptoiLike5) scFv 6257 SEQ ID NO. 756 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6258 SEQ ID NO. 758 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6259 SEQ ID NO. 760 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6260 SEQ ID NO. 762 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6261 SEQ ID NO. 764 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6262 SEQ ID NO. 766 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6263 SEQ ID NO. 768 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6264 SEQ ID NO. 770 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6265 SEQ ID NO. 772 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6266 SEQ ID NO. 774 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6267 SEQ ID NO. 776 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6268 SEQ ID NO. 778 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6269 SEQ ID NO. 780 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6270 SEQ ID NO. 782 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6271 SEQ ID NO. 784 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6272 SEQ ID NO. 786 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6273 SEQ ID NO. 788 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6274 SEQ ID NO. 790 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6275 SEQ ID NO. 792 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6276 SEQ ID NO. 794 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6277 SEQ ID NO. 796 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6278 SEQ ID NO. 798 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6279 SEQ ID NO. 800 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6280 SEQ ID NO. 802 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6281 SEQ ID NO. 804 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6282 SEQ ID NO. 806 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6283 SEQ ID NO. 808 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6284 SEQ ID NO. 810 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6285 SEQ ID NO. 812 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6286 SEQ ID NO. 814 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6287 SEQ ID NO. 816 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6288 SEQ ID NO. 818 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6289 SEQ ID NO. 820 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6290 SEQ ID NO. 822 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6291 SEQ ID NO. 824 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6292 SEQ ID NO. 826 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6293 SEQ ID NO. 828 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6294 SEQ ID NO. 830 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6295 SEQ ID NO. 832 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6296 SEQ ID NO. 834 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6297 SEQ ID NO. 836 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6298 SEQ ID NO. 838 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6299 SEQ ID NO. 840 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6300 SEQ ID NO. 842 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6301 SEQ ID NO. 844 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6302 SEQ ID NO. 846 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6303 SEQ ID NO. 848 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6304 SEQ ID NO. 850 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6305 SEQ ID NO. 852 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6306 SEQ ID NO. 854 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6307 SEQ ID NO. 856 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6308 SEQ ID NO. 858 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6309 SEQ ID NO. 860 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6310 SEQ ID NO. 862 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6311 SEQ ID NO. 864 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6312 SEQ ID NO. 866 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6313 SEQ ID NO. 868 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6314 SEQ ID NO. 870 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6315 SEQ ID NO. 872 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6316 SEQ ID NO. 874 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6317 SEQ ID NO. 876 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6318 SEQ ID NO. 878 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6319 SEQ ID NO. 880 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6320 SEQ ID NO. 882 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6321 SEQ ID NO. 884 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6322 SEQ ID NO. 886 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6323 SEQ ID NO. 888 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6324 SEQ ID NO. 890 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6325 SEQ ID NO. 892 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6326 SEQ ID NO. 894 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6327 SEQ ID NO. 896 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6328 SEQ ID NO. 650 in WO2016090337 FcRL5(FcReceptorLike5) scFv 6329 SEQ ID NO. 678 in WO2016090337 Folate Receptor scFv 6330 SEQ ID NO. 15 in US20170002072A1 FOLRl/CD3sBiSpecific scFv 6331 SEQ ID NO. 90 in WO2014144722A2 GCN4 scFv 6332 SEQ ID NO. 165 in WO2016168773A3 GCN4 scFv 6333 SEQ ID NO. 166 in WO2016168773A3 GCN4 scFv 6334 SEQ ID NO. 167 in WO2016168773A3 GCN4 scFv 6335 SEQ ID NO. 168 in WO2016168773A3 GCN4 scFv 6336 SEQ ID NO. 169 in WO2016168773A3 GCN4 scFv 6337 SEQ ID NO. 170 in WO2016168773A3 GD2 scFv 6338 SEQ ID NO. 19 in WO2016134284 GD2 scFv 6339 SEQ ID NO. 20 in WO2016134284 GD2 scFv 6340 SEQ ID NO. 21 in WO2016134284 GD2 scFv 6341 SEQ ID NO. 7 in WO2015132604 GD2 scFv 6342 SEQ ID NO. 8 in WO2015132604 GPC3 scFv 6343 SEQ ID NO. 1 in WO2016049459 GPC3 scFv 6344 SEQ ID NO. 12 in US20160208015A1 GPC4 scFv 6345 SEQ ID NO. 24 in WO2016049459 GPRC5D scFv 6346 SEQ ID NO. 100 in WO2016090312 GPRC5D scFv 6347 SEQ ID NO. 101 in WO2016090312 GPRC5D scFv 6348 SEQ ID NO. 102 in WO2016090312 GPRC5D scFv 6349 SEQ ID NO. 103 in WO2016090312 GPRC5D scFv 6350 SEQ ID NO. 104 in WO2016090312 GPRC5D scFv 6351 SEQ ID NO. 105 in WO2016090312 GPRC5D scFv 6352 SEQ ID NO. 106 in WO2016090312 GPRC5D scFv 6353 SEQ ID NO. 107 in WO2016090312 GPRC5D scFv 6354 SEQ ID NO. 108 in WO2016090312 GPRC5D scFv 6355 SEQ ID NO. 109 in WO2016090312 GPRC5D scFv 6356 SEQ ID NO. 110 in WO2016090312 GPRC5D scFv 6357 SEQ ID NO. 111 in WO2016090312 GPRC5D scFv 6358 SEQ ID NO. 112 in WO2016090312 GPRC5D scFv 6359 SEQ ID NO. 113 in WO2016090312 GPRC5D scFv 6360 SEQ ID NO. 114 in WO2016090312 GPRC5D scFv 6361 SEQ ID NO. 115 in WO2016090312 GPRC5D scFv 6362 SEQ ID NO. 116 in WO2016090312 GPRC5D scFv 6363 SEQ ID NO. 117 in WO2016090312 GPRC5D scFv 6364 SEQ ID NO. 118 in WO2016090312 GPRC5D scFv 6365 SEQ ID NO. 119 in WO2016090312 GPRC5D scFv 6366 SEQ ID NO. 120 in WO2016090312 GPRC5D scFv 6367 SEQ ID NO. 121 in WO2016090312 GPRC5D scFv 6368 SEQ ID NO. 122 in WO2016090312 GPRC5D scFv 6369 SEQ ID NO. 123 in WO2016090312 GPRC5D scFv 6370 SEQ ID NO. 301 in WO2016090312 GPRC5D scFv 6371 SEQ ID NO. 313 in WO2016090312 GPRC5D scFv 6372 SEQ ID NO. 325 in WO2016090312 GPRC5D scFv 6373 SEQ ID NO. 337 in WO2016090312 GPRC5D scFv 6374 SEQ ID NO. 349 in WO2016090312 GPRC5D scFv 6375 SEQ ID NO. 361 in WO2016090312 GPRC5D scFv 6376 SEQ ID NO. 373 in WO2016090312 GPRC5D scFv 6377 SEQ ID NO. 385 in WO2016090312 HER2/CD3 scFv 6378 SEQ ID N0. 9 in WO2014144722A2 humanCD79b1F10 scFv 6379 SEQ ID NO. 33 in WO2016112870 Human collagen VII scFv 6380 SEQ ID NO. 34 in WO2016112870 Integrin Bivalent scFv 6381 SEQ ID N0. 2 in WO2009070753 Integrin Bivalent scFv 6382 SEQ ID NO. 1 in WO2009070753 Ipilimumab scFv 6383 SEQ ID NO. 39 in US20160208021 Ipilimumab scFv 6384 SEQ ID NO. 39 in WO2016112870 IL4 scFv 6385 SEQ ID NO. 17 in WO2009121847 IL4R scFv 6386 SEQ ID NO. 16 in WO2009121847 Mec/CD3sBispecific scFv 6387 SEQ ID NO. 78 in WO2014144722A2 Mesothelin scFv 6388 SEQ ID NO. 7 WO2015188141 Mesothelin scFv 6389 SEQ ID NO 47 in WO2016090034 Mesothelin scFv 6390 SEQ ID NO in 46 in WO2016090034 Mesothelin scFv 6391 SEQ ID NO in 57 in WO2016090034 Mesothelin scFv 6392 SEQ ID NO. 48 in WO2016090034 Mesothelin scFv 6393 SEQ ID NO. 49 in WO2016090034 Mesothelin scFv 6394 SEQ ID NO. 50 in WO2016090034 Mesothelin scFv 6395 SEQ ID NO. 51 in WO2016090034 Mesothelin scFv 6396 SEQ ID NO. 53 in WO2016090034 Mesothelin scFv 6397 SEQ ID NO. 54 in WO2016090034 Mesothelin scFv 6398 SEQ ID NO. 55 in WO2016090034 Mesothelin scFv 6399 SEQ ID NO. 56 in WO2016090034 Mesothelin scFv 6400 SEQ ID NO. 58 in WO2016090034 Mesothelin scFv 6401 SEQ ID NO. 59 WO2016090034 Mesothelin scFv 6402 SEQ ID NO. 62 in WO2016090034 Mesothelin scFv 6403 SEQ ID NO. 64 in WO2016090034 Mesothelin scFv 6404 SEQ ID NO. 65 in WO2016090034 Mesothelin scFv 6405 SEQ ID NO. 66 in WO2016090034 Mesothelin scFv 6406 SEQ ID NO. 67 in WO2016090034 Mesothelin scFv 6407 SEQ ID NO. 68 in WO2016090034 Mesothelin scFv 6408 SEQ ID NO. 69 in WO2016090034 Mesothelin scFv 6409 SEQ ID NO. 7 WO2015188141 Mesothelin scFv 6410 SEQ ID NO. 70 in WO2016090034 Mesothelin scFv 6411 SEQ ID NO. 52 in WO2016090034 Mesothelin scFv 6412 SEQ ID NO. 60 in WO2016090034 Mesothelin scFv 6413 SEQ ID NO. 61 in WO2016090034 Mesothelin scFv 6414 SEQ ID NO. 63 in WO2016090034 Mesothelin scFv 6415 SEQ ID NO. 10 in WO2013142034 Mesothelin scFv 6416 SEQ ID NO. 11 in WO2013142034 Mesothelin scFv 6417 SEQ ID NO. 12 in WO2013142034 Mesothelin scFv 6418 SEQ ID NO. 11 in WO2013063419 MUC1 scFv 6419 SEQ ID NO. 15 in US20160130357 MUC2 scFv 6420 SEQ ID NO. 17 in US20160130357 MUC3 scFv 6421 SEQ ID NO. 15 in US20160130357 MUC4 scFv 6422 SEQ ID NO. 17 in US20160130357 Nivolumab scFv 6423 SEQ ID NO. 38 in US20160208021 Nivolumab scFv 6424 SEQ ID NO. 38 in WO2016112870 NYBR1 scFv 6425 SEQ ID NO. 21 in US20160333422A1 NYBR1 scFv 6426 SEQ ID NO. 21 in WO2015112830 NYBR1 6427 SEQ ID NO. 18 in WO2015112830 NYBR1 6428 SEQ ID NO. 19 in WO2015112830 O acetylated GD2 ganglioside scFv 6429 SEQ ID NO. 29 in US20150140023 O acetylated GD2 ganglioside scFv 6430 SEQ ID NO. 31 in US20150140023 OX40 scFv 6431 SEQ ID NO. 33 in US20150190506 PD1 scFv 6432 SEQ ID NO. 39 in US20160311917A1 PD1 scFv 6433 SEQ ID NO. 40 in US20160311917A1 PD1 scFv 6434 SEQ ID NO. 41 in US20160311917A1 PD1 scFv 6435 SEQ ID NO. 42 in US20160311917A1 PD1 scFv 6436 SEQ ID NO. 43 in US20160311917A1 PD1 scFv 6437 SEQ ID NO. 44 in US20160311917A1 PD1 scFv 6438 SEQ ID NO. 45 in US20160311917A1 PD1 scFv 6439 SEQ ID NO. 46 in US20160311917A1 PD1 scFv 6440 SEQ ID NO. 47 in US20160311917A1 PD1 scFv 6441 SEQ ID NO. 48 in US20160311917A1 PD1 scFv 6442 SEQ ID NO. 49 in US20160311917A1 PD1 scFv 6443 SEQ ID NO. 50 in US20160311917A1 PD1 scFv 6444 SEQ ID NO. 51 in US20160311917A1 PD1 scFv 6445 SEQ ID NO. 52 in US20160311917A1 PD1 scFv 6446 SEQ ID NO. 53 in US20160311917A1 PD1 scFv 6447 SEQ ID NO. 54 in US20160311917A1 PD1 scFv 6448 SEQ ID NO. 55 in US20160311917A1 PD1 scFv 6449 SEQ ID NO. 56 in US20160311917A1 PD1 scFv 6450 SEQ ID NO. 57 in US20160311917A1 PD1 scFv 6451 SEQ ID NO. 58 in US20160311917A1 PD1 scFv 6452 SEQ ID NO. 59 in US20160311917A1 PD1 scFv 6453 SEQ ID NO. 60 in US20160311917A1 PD1 scFv 6454 SEQ ID NO. 61 in US20160311917A1 PDK1 scFv 6455 SEQ ID NO. 15 in WO2016090365 PDL1 Nanobody 6456 SEQ ID NO. 22 in US20110129458 PDL1 Nanobody 6457 SEQ ID NO. 23 in US20110129458 PDL1 Nanobody 6458 SEQ ID NO. 24 in US20110129458 PDL1 Nanobody 6459 SEQ ID NO. 25 in US20110129458 PDL1 Nanobody 6460 SEQ ID NO. 26 in US20110129458 PDL1 Nanobody 6461 SEQ ID NO. 27 in US20110129458 PDL2 Nanobody 6462 SEQ ID NO. 28 in US20110129458 PDL2 Nanobody 6463 SEQ ID NO. 29 in US20110129458 PDL2 Nanobody 6464 SEQ ID NO. 30 in US20110129458 PDL2 Nanobody 6465 SEQ ID NO. 31 in US20110129458 PDL2 Nanobody 6466 SEQ ID NO. 32 in US20110129458 PDL2 Nanobody 6467 SEQ ID NO. 33 in US20110129458 PRAME scFv 6468 SEQ ID NO. 63 in WO2016191246A2 PRAME scFv 6469 SEQ ID NO. 64 in WO2016191246A2 PRAME scFv 6470 SEQ ID NO. 65 in WO2016191246A2 PRAME scFv 6471 SEQ ID NO. 66 in WO2016191246A2 PRAME scFv 6472 SEQ ID NO. 67 in WO2016191246A2 PRAME scFv 6473 SEQ ID NO. 68 in WO2016191246A2 PRAME scFv 6474 SEQ ID NO. 69 in WO2016191246A2 PSMA scFv 6475 SEQ ID NO. 19 in WO2012145714 PSMA scFv 6476 SEQ ID NO. 21 in WO2012145714 PSMA scFv 6477 SEQ ID NO. 30 in WO2012145714 PSMA scFv 6478 SEQ ID NO. 31 in WO2012145714 PSMA scFv 6479 SEQ ID NO. 34 in WO2012145714 PSMA scFv 6480 SEQ ID NO. 35 in WO2012145714 PSMA Diabody 6481 SEQ ID NO. 12 in WO2011069019 PSMA Diabody 6482 SEQ ID NO. 13 in WO2011069019 PSMA Diabody 6483 SEQ ID NO. 14 in WO2011069019 PSMA Diabody 6484 SEQ ID NO. 15 in WO2011069019 radiation inducible neoantigen scFv 6485 SEQ ID NO 22 in WO2005042780A1 radiation inducible neoantigen scFv 6486 SEQ ID NO 24 in WO2005042780A1 Ranibizuman scFv 6487 SEQ ID NO. 40 in US20160208021 Ranibizuman scFv 6488 SEQ ID NO. 40 in WO2016112870 RAS scFv 6489 SEQ ID NO. 81 in WO2016154047 Rituximab scFv 6490 SEQ ID NO. 36 in US20160208021 Rituximab scFv 6491 SEQ ID NO. 36 in WO2016112870 RORI scFv 6492 SEQ ID NO. 34 in EP3083691A2 RORI scFv 6493 SEQ ID NO. 249 in US20160208018A1 RORI scFv 6494 SEQ ID NO. 250 in US20160208018A1 RORI scFv 6495 SEQ ID NO. 251 in US20160208018A1 RORI scFv 6496 SEQ ID NO. 252 in US20160208018A1 RORI scFv 6497 SEQ ID NO. 253 in US20160208018A1 RORI scFv 6498 SEQ ID NO. 254 in US20160208018A1 RORI scFv 6499 SEQ ID NO. 255 in US20160208018A1 RORI scFv 6500 SEQ ID NO. 256 in US20160208018A1 RORI scFv 6501 SEQ ID NO. 257 in US20160208018A1 RORI scFv 6502 SEQ ID NO. 258 in US20160208018A1 RORI scFv 6503 SEQ ID NO. 259 in US20160208018A1 RORI scFv 6504 SEQ ID NO. 260 in US20160208018A1 RORI scFv 6505 SEQ ID NO. 261 in US20160208018A1 RORI scFv 6506 SEQ ID NO. 262 in US20160208018A1 RORI scFv 6507 SEQ ID NO. 263 in US20160208018A1 RORI scFv 6508 SEQ ID NO. 264 in US20160208018A1 RORI scFv 6509 SEQ ID NO. 265 in US20160208018A1 RORI scFv 6510 SEQ ID NO. 266 in US20160208018A1 RORI scFv 6511 SEQ ID NO. 267 in US20160208018A1 RORI scFv 6512 SEQ ID NO. 268 in US20160208018A1 RORI scFv 6513 SEQ ID NO. 57 in EP3083671A1 RORI scFv 6514 SEQ ID NO. 1 in US20160304619A1 RORI scFv 6515 SEQ ID NO. 2 in US20160304619A1 RORI scFv 6516 SEQ ID NO. 34 in WO2015092024A2 Teplizumab scFv 6517 SEQ ID NO. 42 in WO2016112870 Teplizumab(mutated) scFv 6518 SEQ ID NO. 42 in US20160208021 TOSO scFv 6519 SEQ ID NO. 2 in EP3098237A1 Trastuzumab scFv 6520 SEQ ID NO. 35 in US20160208021 Trastuzumab scFv 6521 SEQ ID NO. 35 in WO2016112870 TRBC1 scFv 6522 SEQ ID NO. 13 in WO2015132598 TRBC1 scFv 6523 SEQ ID NO. 14 in WO2015132598 TRBC1 scFv 6524 SEQ ID NO. 15 in WO2015132598 TRBC1 scFv 6525 SEQ ID NO. 16 in WO2015132598 TRBC1 scFv 6526 SEQ ID NO. 17 in WO2015132598 TRBC1 scFv 6527 SEQ ID NO. 18 in WO2015132598 TRBC1 scFv 6528 SEQ ID NO. 19 in WO2015132598 TRBC1 scFv 6529 SEQ ID NO. 20 in WO2015132598 TRBC1 scFv 6530 SEQ ID NO. 21 in WO2015132598 TRBC1 scFv 6531 SEQ ID NO. 22 in WO2015132598 TRBC1 scFv 6532 SEQ ID NO. 3 in WO2015132598 TRBC2 scFv 6533 SEQ ID NO. 23 in WO2015132598 TRBC2 scFv 6534 SEQ ID NO. 24 in WO2015132598 TRBC2 scFv 6535 SEQ ID NO. 25 in WO2015132598 TRBC2 scFv 6536 SEQ ID NO. 26 in WO2015132598 TRBC2 scFv 6537 SEQ ID NO. 27 in WO2015132598 TRBC2 scFv 6538 SEQ ID NO. 28 in WO2015132598 TRBC2 scFv 6539 SEQ ID NO. 29 in WO2015132598 TRBC2 scFv 6540 SEQ ID NO. 30 in WO2015132598 TRBC2 scFv 6541 SEQ ID NO. 31 in WO2015132598 TRBC2 scFv 6542 SEQ ID NO. 32 in WO2015132598 TSLPR scFv 6543 SEQ ID NO. 1 in US20160311910A1 TSLPR scFv 6544 SEQ ID NO. 2 in US20160311910A1 TSLPR scFv 6545 SEQ ID NO. 1 in WO2015084513 TSLPR scFv 6546 SEQ ID NO. 2 in WO2015084513 VEGF scFv 6547 SEQ ID NO. 168 in US20160090427 VEGF scFv 6548 SEQ ID NO. 169 in US20160090427 VEGF scFv 6549 SEQ ID NO. 170 in US20160090427 VEGF scFv 6550 SEQ ID NO. 171 in US20160090427 VEGF scFv 6551 SEQ ID NO. 172 in US20160090427 VEGF scFv 6552 SEQ ID NO. 173 US20160090427 VEGF scFv 6553 SEQ ID NO. 174 in US20160090427 VEGF scFv 6554 SEQ ID NO. 175 in US20160090427 VEGFR scFv 6555 SEQ ID NO. 498 in US20110177074A1 VEGFR scFv 6556 SEQ ID NO. 500 in US20110177074A1 VEGFR scFv 6557 SEQ ID NO. 502 in US20110177074A1 VEGFR scFv 6558 SEQ ID NO. 504 in US20110177074A1 VEGFR scFv 6559 SEQ ID NO. 506 in US20110177074A1 VEGFR scFv 6560 SEQ ID NO. 508 in US20110177074A1 VEGFR2 scFv 6561 SEQ ID NO. 1 in US20120213783 VEGFR2 scFv 6562 SEQ ID NO. 2 in US20120213783 WT1/HLA Bispecific scFv 6563 SEQ ID NO. 108 in WO2015070061 WT1/HLA Bispecific scFv 6564 SEQ ID NO. 113 in WO2015070061 WT1/HLA Bispecific scFv 6565 SEQ ID NO. 18 in WO2015070061 WT1/HLA Bispecific scFv 6566 SEQ ID NO. 36 in WO2015070061 WT1/HLA Bispecific scFv 6567 SEQ ID NO. 54 in WO2015070061 WT1/HLA Bispecific scFv 6568 SEQ ID NO. 72 in WO2015070061 WT1/HLA Bispecific scFv 6569 SEQ ID NO. 90 in WO2015070061 αfolate receptor(FRα) scFv 6570 SEQ ID NO. 15 in WO2012099973 αfolate receptor(FRα) scFv 6571 SEQ ID NO. 23 in WO2012099973

In one embodiment, the targeting moiety of the CAR may recognize CD19. CD19 is a well-known B cell surface molecule, which upon B cell receptor activation enhances B-cell antigen receptor induced signaling and expansion of B cell populations. CD19 is broadly expressed in both normal and neoplastic B cells. Malignancies derived from B cells such as chronic lymphocytic leukemia, acute lymphocytic leukemia and many non-Hodgkin lymphomas frequently retain CD19 expression. This near universal expression and specificity for a single cell lineage has made CD19 an attractive target for immunotherapies. Human CD19 has 14 exons wherein exon 1-4 encode the extracellular portion of the CD19, exon 5 encodes the transmembrane portion of CD19 and exons 6-14 encode the cytoplasmic tail. In one embodiment, the targeting moiety may comprise scFvs derived from the variable regions of the FMC63 antibody. FMC63 is an IgG2a mouse monoclonal antibody clone specific to the CD19 antigen that reacts with CD19 antigen on cells of the B lineage. The epitope of CD19 recognized by the FMC63 antibody is in exon 2 (Sotillo et al (2015) Cancer Discov; 5(12):1282-95; the contents of which are incorporated by reference in their entirety). In some embodiments, the targeting moiety of the CAR may be derived from the variable regions of other CD19 monoclonal antibody clones including but not limited to 4G7, SJ25C1, CVID3/429, CVID3/155, HIB19, and J3-119.

In some embodiments, the targeting moiety of a CAR may recognize a tumor specific antigen (TSA), for example a cancer neoantigen that is only expressed by tumor cells because of genetic mutations or alterations in transcription which alter protein coding sequences, therefore creating novel, foreign antigens. The genetic changes result from genetic substitution, insertion, deletion or any other genetic changes of a native cognate protein (i.e. a molecule that is expressed in normal cells). In the context of CD19, TSAs may include a transcript variant of human CD19 lacking exon 2 or lacking exon 5-6 or both (see International patent publication No. WO2016061368; the contents of which are incorporated herein by reference in their entirety). Since FMC63 binding epitope is in exon 2, CD19 lacking exon 2 is not recognized by FMC63 antibody. Thus, in some embodiments, the targeting moiety of the CAR may be an FMC63-distinct scFV. As used herein “FMC63-distinct” refers, to an antibody, scFv or a fragment thereof that is immunologically specific and binds to an epitope of the CD19 antigen that is different or unlike the epitope of CD19 antigen that is bound by FMC63. In some instances, targeting moiety may recognize a CD19 antigen lacking exon2. In one embodiment, the targeting moiety recognizes a fragment of CD19 encoded by exon 1, 3 and/or 4. In one example, the targeting moiety recognizes the epitope that bridges the portion of CD19 encoded by exon 1 and the portion of CD19 encoded by exon 3.

Intracellular Signaling Domains

The intracellular domain of a CAR fusion polypeptide, after binding to its target molecule, transmits a signal to the immune effector cell, activating at least one of the normal effector functions of immune effector cells, including cytolytic activity (e.g., cytokine secretion) or helper activity. Therefore, the intracellular domain comprises an “intracellular signaling domain” of a T cell receptor (TCR).

In some aspects, the entire intracellular signaling domain can be employed. In other aspects, a truncated portion of the intracellular signaling domain may be used in place of the intact chain as long as it transduces the effector function signal.

In some embodiments, the intracellular signaling domain of the present invention may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM containing cytoplasmic signaling sequences include those derived from TCR CD3zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In one example, the intracellular signaling domain is a CD3 zeta (CD3) signaling domain.

In some embodiments, the intracellular region of the present invention further comprises one or more costimulatory signaling domains which provide additional signals to the immune effector cells. These costimulatory signaling domains, in combination with the signaling domain can further improve expansion, activation, memory, persistence, and tumor-eradicating efficiency of CAR engineered immune cells (e.g., CART cells). In some cases, the costimulatory signaling region contains 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and/or costimulatory molecules. The costimulatory signaling domain may be the intracellular/cytoplasmic domain of a costimulatory molecule, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function-associated antigen-1), LIGHT, NKG2C, B7-H3. In one example, the costimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another example, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In another example, the co-stimulatory signaling domain may be an intracellular domain of GITR as taught in U.S. Pat. NO.: 9, 175, 308; the contents of which are incorporated herein by reference in its entirety.

In some embodiments, the intracellular region of the present invention may comprise a functional signaling domain from a protein selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation protein (SLAM) such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, SLAMF6, SLAMF7, an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, IL15Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NKD2C SLP76, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, CD270 (HVEM), GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, DAP 10, TRIM, ZAP70, Killer immunoglobulin receptors (KIRs) such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, and KIR2DP1; lectin related NK cell receptors such as Ly49, Ly49A, and Ly49C.

In some embodiments, the intracellular signaling domain of the present invention may contain signaling domains derived from JAK-STAT. In other embodiments, the intracellular signaling domain of the present invention may contain signaling domains derived from DAP-12 (Death associated protein 12) (Topfer et al., Immunol., 2015, 194: 3201-3212; and Wang et al., Cancer Immunol., 2015, 3: 815-826). DAP-12 is a key signal transduction receptor in NK cells. The activating signals mediated by DAP-12 play important roles in triggering NK cell cytotoxicity responses toward certain tumor cells and virally infected cells. The cytoplasmic domain of DAP12 contains an Immunoreceptor Tyrosine-based Activation Motif (ITAM). Accordingly, a CAR containing a DAP12-derived signaling domain may be used for adoptive transfer of NK cells.

In some embodiments, T cells engineered with two or more CARs incorporating distinct co-stimulatory domains and regulated by distinct DD may be used to provide kinetic control of downstream signaling.

In some embodiments, the payload of the invention may be any of the co-stimulatory molecules and/or intracellular domains described herein. In some embodiments, one or more co-stimulatory molecules, each under the control of different SRE may be used in the present invention. SRE regulated co-stimulatory molecules may also be expressed in conjunction with a first-generation CAR, a second-generation CAR, a third-generation CAR, a fourth-generation, or any other CAR design described herein.

In some embodiments, the intracellular domain of the present invention may comprise amino acid sequences of Table 12.

TABLE 12 Intracellular signaling and co-stimulatory domains SEQ Domain Amino Acid Sequence ID NO. 2B4 co-stimulatory domain WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTF 6572 PGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRK RNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS CD27 co-stimulatory domain HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDY 6573 RKPEPACSP CD272 (BTLA1) co- RRHQGKQNELSDTAGREINLVDAKLKSEQTEASTRQNSQ 6574 stimulatory domain VLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVY ASLNHSVIGPNSRLARNVKEAPTEYASICVRS CD272 (BTLA1) co- CCLRRHQGKQNELSDTAGREINLVDAHLKSEQILASTRQ 6575 stimulatory domain NSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKP GIVYASLNHSVIGPNSRLARNVKEAPIEYASICVRS CD28 co-stimulatory FWVLVVVGGVLACYSLLVTVAFIIFWV 6576 CD28 co-stimulatory domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG 6577 CEL CD28 co-stimulatory domain FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD 6578 FAAYRS CD28 co-stimulatory domain RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA 6579 YRS CD28 co-stimulatory domain RSKRSRGGHSDYIVINMTPRRPGPTRKHYQPYAPPRDFA 6580 AYRS CD28 co-stimulatory MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLS 6581 signaling region CKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQ VYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVL VVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM TPRRPGPTRKHYQPYAPPRDFAAYRS CD30 co-stimulatory domain RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLR 6582 SGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQD ASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVG TVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPP LGSCSDVMLSVEEEGKEDPLPTAASGK CD30 co-stimulatory domain RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLR 6583 SGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQD ASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVG TVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPP LGSCSDVMLSVEEEGKEDPLPTAASGK GITR co-stimulatory domain HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERG 6584 ERSAEEKGRLGDLWV HVEM co-stimulatory CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPP 6585 domain DVTTVAVEETIPSFTGRSPNH ICOS co-stimulatory domain TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL 6586 ICOS co-stimulatory CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVT 6587 signaling domain L LAG-3 co-stimulatory region HLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPE 6588 PEPEPEPEPEPEQL OX40 co-stimulatory domain ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTL 6589 AKI OX40 co-stimulatory domain RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 6590 4-1BB intracellular domain KRGRKKLLYIFKQPFMRPVQTIQEEDGCSCRFPEEEEGGC 6591 EL 4-1BB signaling domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG 6592 YEL 4-1BB-CD 3Zeta intracellular TGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR 6593 domain GLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR 4-1BB-Z endodo main fusion KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG 6594 CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR CD127 intracellular domain KRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLD 6595 CQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDV QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSS RSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGIL TLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ CD137 intracellular domain RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE 6596 EEGGCEL CD148 intracellular domain RKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQA 6597 DSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNN VLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIAT QGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCE EYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTS ESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQS PPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGI VYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVD LIYQNTTAMTIYENLAPVTTFGKTNGYIA CD27 intracellular domain QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYR 6598 KPEPACSP CD28 intracellular domain FAAYRS 6599 CD28 signaling chain FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY 6600 MNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 signaling domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA 6601 YRS CD28 signaling domain SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY 6602 RS CD28 signaling domain IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF 6603 WVLVVVGGVLACYSLLVTVAFIIFWRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28, 4-1BB, and/or CD3C RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA 6604 signaling domain YRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR CD28/CD3C AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPS 6605 KPFWVLVVVGGVLACYSLLVTVAFBFWVRSKRSRLLHS DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR CD28-0XZ intracellular RSKRSRLLHSDYNMTPRRPGPTRKHYQPYAPPRDFAAYR 6606 domain SRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD28-4-1BB intracellular MFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIF 6607 domain KQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD28-4-1BB intracellular IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF 6608 domain WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD28-CD3 Zeta intracellular RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA 6609 domain YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR CD28-CD3Zeta intracellular KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR 6610 domain SRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 delta chain intracellular MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSI 6611 signaling domain TWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKD KESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLAL GVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDA QYSHLGGNWARNK CD3 delta chain intracellular MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSI 6612 signaling domain TWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKD KESTVQVHYRTADTQALLRNDQVYQPLRDRDDAQYSHL GGNWARNK CD3 delta chain intracellular DQVYQPLRDRDDAQYSHLGGN 6613 signaling domain CD3 delta intracellular MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSI 6614 domain TWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKD KESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLAL GVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDA QYSHLGGNWARNK CD3 delta intracellular MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSI 6615 domain TWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKD KESTVQVHYRTADTQALLRNDQVYQPLRDRDDAQYSHL GGNWARNK CD3 delta intracellular DQVYQPLRDRDDAQYSHLGGN 6616 domain CD3 epsilon intracellular MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYK 6617 domain VSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGS DEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRA RVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNR KAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRK GQRDLYSGLNQRRI CD3 epsilon intracellular NPDYEPIRKGQRDLYSGLNQR 6618 domain CD3 gamma intracellular MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQED 6619 domain GSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSN AKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATI SGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLP NDQLYQPLKDREDDQYSHLQGNQLRRN CD3 gamma intracellular DQLYQPLKDREDDQYSHLQGN 6620 domain CD3 gamma intracellular DQLYQPLKDREDDQYSHLQGN 6621 domain CD3 gamma intracellular MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQED 6622 domain GSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSN AKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATI SGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLP NDQLYQPLKDREDDQYSHLQGNQLRRN CD3 zeta intmcellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILF 6623 domain IYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR CD3 zeta intmcellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILF 6624 domain IYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR CD3 zeta intmcellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILF 6625 domain IYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR CD3 zeta intmcellular NQLYNELNLGRREEYDVLDKR 6626 domain CD3 zeta domain 2 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR 6627 (NM_000734.3) RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular DGLYQGLSTATKDTYDALHMQ 6628 domain CD3 zeta intmcellular RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKR 6629 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR 6630 domain RGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD 6631 domain KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R CD3 zeta intmcellular RVKFSRSADAPAYQQGEYDVLDKRRGRDPEMGGKPRRK 6632 domain NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEVDVLDKR 6633 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLVGIVLTL 6634 domain LVCRLKIQVRKAAITSYEKSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAVSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR CD3 zeta intmcellular LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK 6635 domain RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR 6636 domain RGRDPEMGGKPQRRKNPQEGLY CD3 zeta intmcellular LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK 6637 domain RRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R CD3 zeta intmcellular RRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK 6638 domain RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular NQLYNELNLGRREEYDVLDKR 6639 domain CD3 zeta intmcellular EGLYNELQKDKMAEAYSEIGMK 6640 domain CD3 zeta intmcellular DGLYQGLSTATKDTYDALHMQ 6641 domain CD3 zeta intmcellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR 6642 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKR 6643 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR 6644 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALP CD3 zeta intmcellular DPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQG 6645 domain QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR CD3 zeta intmcellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILF 6646 domain IYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR CD40 intracellular domain RSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 6647 CD79A intracellular domain MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKV 6648 PASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYT WPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESY QQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCA VVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLD DCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP CD79A intracellular domain MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKV 6649 PASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYT WPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLF CAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLN LDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP CD79A intracellular domain MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKV 6650 PASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYT WPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESY QQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCA VVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLD DCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP CD79A intracellular domain ENLYEGLNLDDCSMYEDISRG 6651 CD8 intracellular domain FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA 6652 GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNR CD8 intracellular domain FVPVFLPAKPITTPAPRPPTPAPTIASQPLSLRPEACRPAAG 6653 GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH RNR CD8a intracellular domain PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL 6654 DFACDI CTLA4 intracellular domain AVSLSKMLKKRSPLTTGVFVKMAPTEAECEKQFQPYFIPI 6655 N CTLA4 intracellular domain AVSLSKMLKKRSPLTTGTPRRPECEKQFQPYAPP 6656 RDFAAYRS DAP10 intracellular domain RPRRSPAQDGKVYINMPGRG 6657 DAP12 intracellular domain MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 6658 GVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAA TRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK DAP12 intracellular domain MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 6659 GVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATR KQRITETESPYQELQGQRSDVYSDLNTQRPYYK DAP12 intracellular domain MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGD 6660 LVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP YQELQGQRSDVYSDLNTQRPYYK DAP12 intracellular domain MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGD 6661 LVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESPY QELQGQRSDVYSDLNTQRPYYK DAP12 intracellular domain MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 6662 GVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAA TRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK DAP12 intracellular domain MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 6663 GVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATR KQRITETESPYQELQGQRSDVYSDLNTQRPYYK DAP12 intracellular domain MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGD 6664 LVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP YQELQGQRSDVYSDLNTQRPYYK DAP12 intracellular domain MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGD 6665 LVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESPY QELQGQRSDVYSDLNTQRPYYK DAP12 intracellular domain ESPYQELQGQRSDVYSDLNTQ 6666 DAP12 intracellular domain ESPYQELQGQRSDVYSDLNTQ 6667 GITR intracellular domain RSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEE 6668 KGRLGDLWV ICOS intracellular domain TKKKYSSSVHDPNGEFMFMRAVNTAKKSRLTDVTL 6669 IL 15Ra intracellular domain KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 6670 OX40-CD3 Zeta intracellular RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVK 6671 domain FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR ZAP70 intracellular domain MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQC 6672 LRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHC GPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCL RDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTA HERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQ GTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQ LVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAH PSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTS VYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVR QGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIM HQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVG KREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARN VLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLK WYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKK MKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKW EDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAAC A CD28 intracellular domain MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLS 6673 CKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQ VYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVL VVVGGVLACYSLLVTVAFIIFWVR 4-1BB intracellular domain MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDN 6674 NRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECS STSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC KDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERD VVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTST ALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQE EDG Fc epsilon Receptor I gamma MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTL 6675 chain intmcellular domain LYCRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLK HEKPPQ Fc epsilon Receptor I gamma DGVYTGLSTRNQETYETLKHE 6676 chain intmcellular domain Fc epsilon Receptor I gamma DPKLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKS 6677 chain intmcellular domain DGVYTGLSTRNQETYETLKHEKPPQ Fc epsilon Receptor I gamma DGVYTGLSTRNQETYETLKHE 6678 chain intmcellular domain

Transmembrane Domains

In some embodiments, the CAR of the present invention may comprise a transmembrane domain. As used herein, the term “Transmembrane domain(TM)” refers broadly to an amino acid sequence of about 15 residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domain of the present invention may be derived either from a natural or from a synthetic source. The transmembrane domain of a CAR may be derived from any naturally membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, or CD154.

Alternatively, the transmembrane domain of the present invention may be synthetic. In some aspects, the synthetic sequence may comprise predominantly hydrophobic residues such as leucine and valine.

In some embodiments, the transmembrane domain of the present invention may be selected from the group consisting of a CD8a transmembrane domain, a CD4 transmembrane domain, a CD 28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and a human IgG4 Fc region. As non-limiting examples, the transmembrane domain may be a CTLA-4 transmembrane domain comprising the amino acid sequences of SEQ ID NOs.: 1-5 of International Patent Publication NO.: WO2014/100385; and a PD-1 transmembrane domain comprising the amino acid sequences of SEQ ID NOs.: 6-8 of International Patent Publication NO.: WO2014100385; the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the CAR of the present invention may comprise an optional hinge region (also called spacer). A hinge sequence is a short sequence of amino acids that facilitates flexibility of the extracellular targeting domain that moves the target binding domain away from the effector cell surface to enable proper cell/cell contact, target binding and effector cell activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge sequence may be positioned between the targeting moiety and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. The hinge sequence may be derived from all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge, the extracellular regions of type 1 membrane proteins such as CD8a CD4, CD28 and CD7, which may be a wild type sequence or a derivative. Some hinge regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. In certain embodiments, the hinge region may be modified from an IgG1, IgG2, IgG3, or IgG4 that includes one or more amino acid residues, for example, 1, 2, 3, 4 or 5 residues, substituted with an amino acid residue different from that present in an unmodified hinge. Table 13 provides various transmembrane regions that can be used in the CARs described herein.

TABLE 13 Transmembrane domains SEQ ID Transmembrane domain Amino Acid Sequence NO. CD8 Transmembrane TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 6679 domain FACDI 4-1BB Transmembrane IISFFLALTSTALLFLLFFLTLRFSVVKRGR 6680 domain 4-1BB Transmembrane IISFFLALTSTALLFLLFFLTLRFSVV 6681 domain CD134 (OX40) VAAILGLGLVLGLLGPLAILLALYLL 6682 Transmembrane domain CD148 Transmembrane AVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKP 6683 and intracellular domain KKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQ PKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYI NANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVY AIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIV LPEVVTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTD LLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDR LIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQ CVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTN GYIA CD148 Transmembrane AVFGCIFGALVIVTVGGFIFW 6684 domain CD2 Transmembrane KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSD 6685 domain KKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDI YKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLT CEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKF KCTAGNKVSKESSVEPVSCPEKGLD CD28 Transmembrane and IEVMYPPPYLDNEKSNGTITHVKGKHLCPSPLFPGPSKPFW 6686 intracellular domain VLVVVGGVLACYSLLVTVAHIFWVRSKRSRLLHSDYMN MTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 Transmembrane FWVLVVVGGVLACYSLLVTVAFIIFWV 6687 domain CD28 Transmembrane IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW 6688 domain VLVVVGGVLACYSLLVTVAFIIFWV CD28 Transmembrane IFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRR 6689 domain CD28 Transmembrane FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY 6690 domain MNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 Transmembrane MFWVLVVVGGVLACYSLLVTVAFIIFWV 6691 domain CD28 Transmembrane FWVLVVVGGVLACYSLLVTVAFHFWV 6692 domain CD28 Transmembrane MFWVLVVVGGVLACYSGGVTVAFIIFWV 6693 domain CD28 Transmembrane WVLVVVGGVLACYSLLVTVAFIIFWV 6694 domain CD28 Transmembrane PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY 6695 domain MNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 Transmembrane FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY 6696 domain and CD28 and MNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA CD3 Zeta intracellular PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK domain PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR CD28 Transmembrane FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY 6697 domain and CD28, OX40, MNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAH and CD3 Zeta intmcellular KPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQ domain QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR CD28 Transmembrane FWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAP 6698 domain and CD3 Zeta AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP intracellular domain RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR CD28 transmembrane-CD3 AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK 6699 zeta signaling domain PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY (“28z”) MNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR CD3 zeta Transmembmne LCYLLDGILFIYGVILTALFLRV 6700 domain CD3 zeta Transmembmne MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFI 6701 domain YGVILTALFL CD3 zeta Transmembmne LCYLLDGILFIYGVILTALFL 6702 domain CD4 Transmembrane ALIVLGGVAGLLLFIGLGIFFCVRC 6703 domain CD4 Transmembrane MALIVLGGVAGLLLFIGLGIFF 6704 domain CD45 Transmembrane and ALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELV 6705 intracellular domain ERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSI PRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEING DAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMI WEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGD VVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTS WPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIWHCSAGVG RTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMV QVEAQYILIHQALVEYNQFGEIEVNLSELHPYLHNMKKR DPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSN VIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSK YINASFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVK VIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDK SSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEP KELISMIQWKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQ TGIFCALLNLLESAETEEWDIFQWKALRKARPGMVSTFEQ YQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVK QDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNG PASPALNQGS CD62L Transmembrane PLFIPVAVMVTAFSGLAFIIWLA 6706 domain CD7 Transmembrane ALPAALAVISFLLGLGLGVACVLA 6707 domain CD8 Transmembrane MALPVTALLLPLALLLHAARP 6708 domain CD8 Transmembrane AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP 6709 domain and CD28 AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC signaling domain NHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR CD8 transmembmne AAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR 6710 domain-CD137 (4-1BB) GLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI signaling domain and CD3 FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA zeta signaling domain DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG (“BBz”) GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR CD8a Transmembrane FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG 6711 domain GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHR N CD8a Transmembrane IWAPLAGTCGVLLLSLVITLYC 6712 domain CD8a Transmembrane IYIWAPLAGTCGVLLLSLVITLYC 6713 domain CD8a Transmembrane IYIWAPLAGTCGVLLLSLVITLYCR 6714 domain CD8a Transmembrane IYIWAPLAGTCGVLLLSLVITLVCR 6715 domain CD8a Transmembrane IYIWAPLAGTCGVLLLSLVIT 6716 domain CD8a Transmembrane IYIWAPLAGTCGVLLLSLVITLY 6717 domain CD8b Transmembrane LGLLVAGVLVLLVSLGVAIHLCC 6718 domain EpoR Transmembrane APVGLVARLADESGHVVLRWLPPPETPMTSHIRYEVDVS 6719 domain AGNGAGSVQRVEILEGRIECVLSNLRGRTRYTFAVRARM AEPSFGGFWSAWSEPVSLLTPSD FcERI a-Transmembrane MAPAMESPTLLCVALLFFAPDGVLAVPQKPKVSLNPPWN 6720 domain RIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETNSSLNI VNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASA EVVMEGQPLFLRCHGWRNWDVYKVIYYKDGEALKYWY ENHNISITNATVEDSGTYYCTGKVWQLDYESEPLNITVIKA PREKYWLQFFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRT RKGFRLLNPHPKPNPKNN FceRIa Transmembrane DIFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRTRKGFRLL 6721 domain NPHPKPNPKNNR GITR Transmembrane PLGWLTVVLLAVAACVLLLTSAQLGLHIWQL 6722 domain Her2 Transmembrane SIISAVVGILLVVVLGVVFGILII 6723 domain Her2 Transmembrane CHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCP 6724 domain SGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKG CPAEQRASPLTSIISAVVGILLVVVLGVVFGILI ICOS Transmembrane FWLPIGCAAFVVVCILGCILI 6725 domain IgG1 Transmembrane EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 6726 domain EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD OX40 Transmembrane VAAILGLGLVLGLLGPLAILL 6727 domain Transmembrane domain IYIWAPLAGTCGVLLLSLVITLYC 6728 Transmembrane domain IYIWAPLAGTCGVLLLSLVITLYC 6729

Hinge region sequences useful in the present invention are provided in Table 14.

TABLE 14 Hinge regions SEQ ID Hinge Domain Amino Acid Sequence NO. Hinge DKTHT 6730 Hinge CPPC 6731 Hinge CPEPKSCDTPPPCPR 6732 Hinge ELKTPLGDTTHT 6733 Hinge KSCDKTHTCP 6734 Hinge KCCVDCP 6735 C233P Hinge KYGPPCP 6736 C233S Hinge VEPKSPDKTHTCPPCP 6737 CD28 Hinge LDPKSSDKTHTCPPCP 6738 CD8a Hinge IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP 6739 CD8a Hinge GGAVHTRGLDFA 6740 CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 6741 FACD CD8a Hinge AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR 6742 GLDFACD CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 6743 FACD CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 6744 FACD CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 6745 FACDEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDT CD8a Hinge PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT 6746 RGLDFACDIY CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 6747 FACDIYIWAPLAGTCGVLLLSLVITLYC CD8a Hinge TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF 6748 ACD Delta5 Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 6749 FACDIY EpoR Hinge LDKTHTCPPCP 6750 FCRIIα Hinge APVGLVARLADESGHVVLRWLPPPETPMTSHIRYEVDVS 6751 AGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARM AEPSFGGFWSAWSEPVSLLTPSD FcγRIIIα Hinge GLAVSTISSFFPPGYQ 6752 Hinge GLAVSTISSFFPPGYQ 6753 Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 6754 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH Hinge YVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPK 6755 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKKDPK Hinge KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG 6756 LDFA Hinge LEPKSCDKTHTCPPCP 6757 Hinge KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG 6758 LD Hinge EPKSCDKTHTCPPCP 6759 Hinge ELKTPLGDTHTCPRCP 6760 Hinge EPKSCDTPPPCPRCP 6761 Hinge ESKYGPPCPSCP 6762 Hinge (CH2- ERKCCVECPPCP 6763 CH3) Hinge (CH3) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 6764 CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgD Hinge ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCL 6765 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 6766 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 6767 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLHPSL PPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEV SGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWA WSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSY VTDH IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 6768 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMVVLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH IgD Hinge ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEE 6769 KKKEKEKEEQEERETKTP IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 6770 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 6771 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 6772 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPQPGSTTFWA WSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSY VTDH IgG1 (CH2 CH3) RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 6773 Hinge domain GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH IgG1 (CH2 CH3) AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIART 6774 Hinge domain PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD IgG1 Hinge AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIART 6775 PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD IgG1 Hinge AEPKSPDKTHTCPPCPKDPK 6776 IgG1 Hinge EPKSCDKTHTCPPCP 6777 IgG1 Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 6778 EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEVKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD IgG1 Hinge SVFLFPPKPKDTL 6779 IgG1 Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 6780 EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG1 Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 6781 EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK IgG1 Hinge VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD 6782 (CH2CH3 VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV domain) SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK IgG2 Hinge DPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIA 6783 RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKK IgG3 Hinge ERKCCVECPPCP 6784 IgG3 Hinge ELKTPLGDTTHTCPRCP 6785 IgG4 (CH2 and ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC 6786 CH3) PRCPEPKSCDTPPPCPRCP IgG4 (CH2 and ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 6787 CH3) CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKM IgG4 Hinge ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT 6788 CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKM IgG4 Hinge SPNMVPHAHHAQ 6789 IgG4 Hinge GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE 6790 WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4 Hinge ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEM 6791 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK IgG4 Hinge ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT 6792 CVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFVPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4 Hinge ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT 6793 CVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFVPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4 Hinge ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 6794 CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKM IgG4 Hinge ESKYGPPCPPCP 6795 IgG4 Hinge ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCL 6796 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4 Hinge ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 6797 CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4 Hinge ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 6798 CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4 Hinge YGPPCPPCP 6799 IgG4 Hinge KYGPPCPPCP 6800 IgG4 Hinge EVVKYGPPCPPCP 6801 IgG4 Hinge and ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 6802 Linker CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDLSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG1 Hinge ESKYGPPCPPCPGGGSSGGGSG 6803 IgG1 Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 6804 EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG1 Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 6805 EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG1 Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 6806 EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, hinge domains and the transmembrane domains may be paired. The hinge domain may be present at the N terminus of the transmembrane domain, at C terminus of the transmembrane domain or within the transmembrane domain. Hinge and transmembrane region sequences which may be useful in the present invention are provided in Table 15.

TABLE 15 Paired Hinge and Transmembrane regions SEQ ID Hinge Domain Amino Acid Sequence NO. CD8a TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF 6807 Transmembmne ACDIYIWAPLAGTCGVLLLSLVITLYC and Hinge CD8a DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQK 6808 Transmembmne PGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTE and Hinge DVATYYCQQYWSTPFTFGSGTKLEIKGGGGSGGGGSGGG GSQVQLKESGPGLVAPSQSLSITSTVSGFSLSRYSVHWVR QPPGKGLEWLGMIWGGGSTDYNSALKSRLSISKDNSKSQ VFLKMNSLQTDDTAMYYCARNEGDTTAGTWFAYWGQG TLVTVSS CD8a ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLS 6809 Transmembmne LRPEACRPAAGGAVHTRGLD and Hinge CD8a TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 6810 Transmembmne FACDIYIWAPLAGTCGVLLLSLVITLY and Hinge CD8a KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG 6811 Transmembmne LDFACDIYIWAPLAGTCGVLLLSLVITLY and Hinge

In some embodiments, the CAR of the present invention may comprise one or more linkers between any of the domains of the CAR. The linker may be between 1-30 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In other embodiments, the linker may be flexible.

In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains of the present invention may be constructed in a single fusion polypeptide. The fusion polypeptide may be the payload of an effector module of the invention. In some embodiments, more than one CAR fusion polypeptides may be included in an effector module, for example, two, three or more CARs may be included in the effector module under the control of a single SRE (e.g., a DD). Representative effector modules comprising the CAR payload are illustrated in FIGS. 2-6 .

In some embodiments, payloads of the present invention may comprise the CAR constructs including the extracellular targeting domain, transmembrane domain and intracellular signaling domains that are taught in the art, for example, a CAR targeting mesothelin (U.S. Pat. NOs.: 9,272,002 and 9, 359,447); EGFRvIII specific CARs in U.S. Pat. No. 9,266,960; anti-TAG CARs in U.S. Pat. No. 9,233,125; CD19 CARs in US Patent publication NO.: 2016014533; CD19 CAR having the amino acid sequence of SEQ ID NO.: 24 of US Pat. NO.: 9, 328, 156; CD19 CARs in U.S. Pat. Nos. 8,911,993; 8,975,071; 9,101,584; 9,102,760; and 9,102,761; BCMA (CD269) specific CARs disclosed in International patent publication NOs: WO2016/014565 (SEQ ID Nos.: 109-113 and 213 to 233) and WO2016/014789; CLL-1 (C-type lectin-like molecule 1) CARs comprising the amino acid sequences of SEQ ID NOs: 99, 96, 100, 101, 102, 91, 92, 93, 94, 95, 97, 98, 103, and 197 disclosed in International patent publication NO.: WO2016014535; CD33 specific CARs comprising the amino acid sequences of SEQ ID NOs: 48-56 in International patent publication NO.: WO2016014576; CD33 specific CARs comprising the amino acid sequences of SEQ ID NOs: 19-22, 27-30 and 35-38 in International patent publication NO.: WO2015150526; CD37 specific CARs encoded by the nucleic acids of SEQ ID NOs: 1-5 in US patent publication NO.: US20150329640; GPC3 CAR (International patent publication NO.: WO2016036973), GFR alpha 4 CARs having the amino acid sequences of SEQ ID NOs: 85, 86, 90, 92, 94, 96, 98, 100, 102, and 104 in International patent publication NO.: WO2016025880; CD123 CARs comprising the amino acid sequences of SEQ ID NO: 98, 99, 100 and 101 in International patent publication NOs: WO2016028896; CD123 specific multi-chain CARs in International patent publication NO: WO2015193406; CD123 CARS comprising the amino acid sequences of SEQ ID NO.: 160, 171, 188-197 in International patent publication NO: WO2016/120220; ROR-1 specific CARs comprising the amino acid sequences of SEQ ID NOs: 93, 95 and 117 in International patent publication NO.: WO2016/016344; ROR-1 specific multi-chain CARs in International patent publication NO.: WO2016/016343; trophoblast glycoprotein (5T4, TPBG) specific CARs comprising the amino acid sequences of SEQ ID NOs: 21, 27, 33, 39, 23, 29, 34, 41, 19, 25, 31, 37, 20, 26, 32, 38, 22, 28, 34, 40, 24, 30, 36 and 42 in International patent publication NO.: WO2016034666; EGFRvIII specific CARs comprising the amino acid sequences of SEQ ID NOs: 15, 17, 24, 25, 26 and 27 in International patent publication NO.: WO2016016341; a TEM 8 CAR comprising the amino acid sequence of SEQ ID NO: 1 in International patent publication NO.: WO2014164544, a TEM1 CAR comprising the amino acid sequence of SEQ ID NO:2 in International patent publication NO.: WO2014164544; GPC-3 CAR having the amino acid sequences of SEQ ID NOs: 3 and 26 in International patent publication NO.: WO2016/049459; a chondroitin sulfate proteoglycan-4 (CSPG4) CAR in International patent publication NO.: WO2015/080981; Kappa/lambda CARs in International patent publication NO.: WO2015/164739; GD2 CAR in International patent publication NO.: WO2016/134284; CLL1 CARs in International patent publication NO.: WO2016120218; CLL1 multi-subunit CARs in International patent publication NO.: WO2016120219; Hsp70 CARs in International patent publication NO.: WO2016120217; mAb-driven CARs in International patent publication NO.: WO2016120216; the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the CAR constructs of the present invention may include CAIX (carboxy-anhydrase-IX (CAIX) specific CAR (Lamers et al., Biochem Soc Trans, 2016, 44(3): 951-959), HIV-1 specific CAR (Ali et al., J Virol., 2016, May 25, pii: JVI.00805-16), CD20 specific CAR (Rufener et al., Cancer Immunol. Res., 2016, 4(6): 509-519), a CD20/CD19 bispecific CAR (Zah et al., Cancer Immunol Res., 2016, 4(6): 498-508), a CD22/CD19 CAR (International Publication No: WO2016/149578), a CD138/BCMA bi-specific CAR (International Publication No: WO2016/130598) an EGFR specific CARs and anti EGFR viii specific CAR; the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the CAR sequences may be selected from Table 16.

TABLE 16 CAR sequences Description SEQ ID NO Source CD70 CAR 6812 SEQ ID NO. 99 in WO2015121454 Acid/Base Leucine zipper zip CAR 6813 SEQ ID NO. 34 in WO2016124930 Acid/Base Leucine zipper zip CAR 6814 SEQ ID NO. 35 in WO2016124930 ALK CAR 6815 SEQ ID NO. 43 in WO2015069922 ALK CAR 6816 SEQ ID NO. 44 in WO2015069922 ALK CAR 6817 SEQ ID NO. 45 in WO2015069922 ALK CAR 6818 SEQ ID NO. 46 in WO2015069922 ALK CAR 6819 SEQ ID NO. 47 in WO2015069922 ALK CAR 6820 SEQ ID NO. 48 in WO2015069922 ALK CAR 6821 SEQ ID NO. 49 in WO2015069922 ALK CAR 6822 SEQ ID NO. 50 in WO2015069922 ALK CAR 6823 SEQ ID NO. 51 in WO2015069922 ALK CAR 6824 SEQ ID NO. 52 in WO2015069922 ALK CAR 6825 SEQ ID NO. 53 in WO2015069922 ALK CAR 6826 SEQ ID NO. 54 in WO2015069922 ALK CAR 6827 SEQ ID NO. 55 in WO2015069922 ALK CAR 6828 SEQ ID NO. 56 in WO2015069922 ALK CAR 6829 SEQ ID NO. 57 in WO2015069922 ALK CAR 6830 SEQ ID NO. 58 in WO2015069922 ALK CAR 6831 SEQ ID NO. 59 in WO2015069922 ALK CAR 6832 SEQ ID NO. 60 in WO2015069922 ALK CAR 6833 SEQ ID NO. 61 in WO2015069922 ALK CAR 6834 SEQ ID NO. 62 in WO2015069922 ALK CAR 6835 SEQ ID NO. 63 in WO2015069922 ALK CAR 6836 SEQ ID NO. 64 in WO2015069922 ALK CAR 6837 SEQ ID NO. 65 in WO2015069922 ALK CAR 6838 SEQ ID NO. 66 in WO2015069922 ALK CAR 6839 SEQ ID NO. 67 in WO2015069922 ALK CAR 6840 SEQ ID NO. 68 in WO2015069922 ALK CAR 6841 SEQ ID NO. 69 in WO2015069922 ALK CAR 6842 SEQ ID NO. 70 in WO2015069922 ALK CAR 6843 SEQ ID NO. 71 in WO2015069922 ALK CAR 6844 SEQ ID NO. 72 in WO2015069922 ALK CAR 6845 SEQ ID NO. 73 in WO2015069922 ALK CAR 6846 SEQ ID NO. 74 in WO2015069922 ALK CAR 6847 SEQ ID NO. 75 in WO2015069922 ALK CAR 6848 SEQ ID NO. 76 in WO2015069922 ALK CAR 6849 SEQ ID NO. 77 in WO2015069922 ALK CAR 6850 SEQ ID NO. 78 in WO2015069922 ALK CAR 6851 SEQ ID NO. 79 in WO2015069922 ALK CAR 6852 SEQ ID NO. 80 in WO2015069922 ALK CAR 6853 SEQ ID NO. 81 in WO2015069922 ALK CAR 6854 SEQ ID NO. 82 in WO2015069922 ALK CAR 6855 SEQ ID NO. 83 in WO2015069922 ALK CAR 6856 SEQ ID NO. 84 in WO2015069922 ALK CAR 6857 SEQ ID NO. 85 in WO2015069922 ALK CAR 6858 SEQ ID NO. 86 in WO2015069922 ALK CAR 6859 SEQ ID NO. 87 in WO2015069922 ALK CAR 6860 SEQ ID NO. 88 in WO2015069922 ALK CAR 6861 SEQ ID NO. 89 in WO2015069922 ALK CAR 6862 SEQ ID NO. 90 in WO2015069922 APRIL IgG1 hinge based CAR 6863 SEQ ID NO: 53 in US20160296562A1 APRIL Fcpvaa based CAR 6864 SEQ ID NO: 52 in US20160296562A1 BCMA CAR 6865 SEQ ID NO: 180 in WO2016168595A1 BCMA CAR 6866 SEQ ID NO: 180 in WO2016168595A1 BCMA CAR 6867 SEQ ID NO: 162 in WO2016168595A1 BCMA CAR 6868 SEQ ID NO: 168 in WO2016168595A1 BCMA CAR 6869 SEQ ID NO: 174 in WO2016168595A1 BCMA CAR 6870 SEQ ID NO: 144 in WO2016168595A1 BCMA CAR 6871 SEQ ID NO. 150 in WO2016168595A1 BCMA CAR 6872 SEQ ID NO: 186 in WO2016168595A1 BCMA CAR 6873 SEQ ID NO: 192 in WO2016168595A1 BCMA CAR 6874 SEQ ID NO: 198 in WO2016168595A1 BCMA CAR 6875 SEQ ID NO. 204 in WO2016168595A1 BCMA CAR 6876 SEQ ID NO: 210 in WO2016168595A1 BCMA CAR 6877 SEQ ID NO: 156 in WO2016168595A1 BCMA CAR 6878 SEQ ID NO: 216 in WO2016168595A1 BCMA CAR 6879 SEQ ID NO: 222 in WO2016168595A1 BCMA CAR 6880 SEQ ID NO: 228 in WO2016168595A1 BCMA CAR 6881 SEQ ID NO: 234 in WO2016168595A1 BCMA CAR 6882 SEQ ID NO: 240 in WO2016168595A1 BCMA CAR 6883 SEQ ID NO: 246 in WO2016168595A1 BCMA CAR 6884 SEQ ID NO: 252 in WO2016168595A1 BCMA CAR 6885 SEQ ID NO: 258 in WO2016168595A1 BCMA CAR 6886 SEQ ID NO: 264 in WO2016168595A1 BCMA CAR 6887 SEQ ID NO: 270 in WO2016168595A1 BCMA CAR 6888 SEQ ID NO: 276 in WO2016168595A1 BCMA CAR 6889 SEQ ID NO. 48 in WO2015158671A1 BCMA CAR 6890 SEQ ID NO. 49 in WO2015158671A1 BCMA CAR 6891 SEQ ID NO. 50 in WO2015158671A1 BCMA CAR 6892 SEQ ID NO. 51 in WO2015158671A1 BCMA CAR 6893 SEQ ID NO. 52 in WO2015158671A1 BCMA CAR 6894 SEQ ID NO. 53 in WO2015158671A1 BCMA CAR 6895 SEQ ID NO. 54 in WO2015158671A1 BCMA CAR 6896 SEQ ID NO. 55 in WO2015158671A1 BCMA CAR 6897 SEQ ID NO. 56 in WO2015158671A1 BCMA CAR 6898 SEQ ID NO. 57 in WO2015158671A1 BCMA CAR 6899 SEQ ID NO. 58 in WO2015158671A1 BCMA CAR 6900 SEQ ID NO. 59 in WO2015158671A1 BCMA CAR 6901 SEQ ID NO. 19 in WO2015158671A1 BCMA CAR 6902 SEQ ID NO. 20 in WO2015158671A1 BCMA CAR 6903 SEQ ID NO. 21 in WO2015158671A1 BCMA CAR 6904 SEQ ID NO. 22 in WO2015158671A1 BCMA CAR 6905 SEQ ID NO. 23 in WO2015158671A1 BCMA CAR 6906 SEQ ID NO. 24 in WO2015158671A1 BCMA CAR 6907 SEQ ID NO. 25 in WO2015158671A1 BCMA CAR 6908 SEQ ID NO. 26 in WO2015158671A1 BCMA CAR 6909 SEQ ID NO. 27 in WO2015158671A1 BCMA CAR 6910 SEQ ID NO. 28 in WO2015158671A1 BCMA CAR 6911 SEQ ID NO. 29 in WO2015158671A1 BCMA CAR 6912 SEQ ID NO. 30 in WO2015158671A1 BCMA CAR 6913 SEQ ID NO. 31 in WO2015158671A1 BCMA CAR 6914 SEQ ID NO. 32 in WO2015158671A1 BCMA CAR 6915 SEQ ID NO. 33 in WO2015158671A1 BCMA CAR 6916 SEQ ID NO. 34 in WO2015158671A1 BCMA CAR 6917 SEQ ID NO. 35 in WO2015158671A1 BCMA CAR 6918 SEQ ID NO. 36 in WO2015158671A1 BCMA CAR 6919 SEQ ID NO. 37 in WO2015158671A1 BCMA CAR 6920 SEQ ID NO. 38 in WO2015158671A1 BCMA CAR 6921 SEQ ID NO. 39 in WO2015158671A1 BCMA CAR 6922 SEQ ID NO. 40 in WO2015158671A1 BCMA CAR 6923 SEQ ID NO. 41 in WO2015158671A1 BCMA CAR 6924 SEQ ID NO. 42 in WO2015158671A1 BCMA CAR 6925 SEQ ID NO: 330 in WO2016168595A1 BCMA CAR 6926 SEQ ID NO: 282 in WO2016168595A1 BCMA CAR 6927 SEQ ID NO: 300 in WO2016168595A1 BCMA CAR 6928 SEQ ID NO: 306 in WO2016168595A1 BCMA CAR 6929 SEQ ID NO. 336 in WO2016168595A1 BCMA CAR 6930 SEQ ID NO: 354 in WO2016168595A1 BCMA CAR 6931 SEQ ID NO: 288 in WO2016168595A1 BCMA CAR 6932 SEQ ID NO: 312 in WO2016168595A1 BCMA CAR 6933 SEQ ID NO: 294 in WO2016168595A1 BCMA CAR 6934 SEQ ID NO. 342 in WO2016168595A1 BCMA CAR 6935 SEQ ID NO. 324 in WO2016168595A1 BCMA CAR 6936 SEQ ID NO: 318 in WO2016168595A1 BCMA CAR 6937 SEQ ID NO: 348 in WO2016168595A1 BCMA CAR 6938 SEQ ID NO. 124 (WO2016014565) BCMA CAR 6939 SEQ ID NO. 114 (WO2016014565) BCMA CAR 6940 SEQ ID NO. 115 (WO2016014565) BCMA CAR 6941 SEQ ID NO. 116 (WO2016014565) BCMA CAR 6942 SEQ ID NO. 117 (WO2016014565) BCMA CAR 6943 SEQ ID NO. 118 (WO2016014565) BCMA CAR 6944 SEQ ID NO. 119 (WO2016014565) BCMA CAR 6945 SEQ ID NO. 120 (WO2016014565) BCMA CAR 6946 SEQ ID NO. 121 (WO2016014565) BCMA CAR 6947 SEQ ID NO. 122 (WO2016014565) BCMA CAR 6948 SEQ ID NO. 123 (WO2016014565) BCMA CAR 6949 SEQ ID NO. 125 (WO2016014565) BCMA CAR 6950 SEQ ID NO. 126 (WO2016014565) BCMA CAR 6951 SEQ ID NO. 127 (WO2016014565) BCMA CAR 6952 SEQ ID NO. 128 (WO2016014565) BCMA CAR 6953 SEQ ID NO. 234 (WO2016014565) BCMA CAR 6954 SEQ ID NO. 235 (WO2016014565) BCMA CAR 6955 SEQ ID NO. 236 (WO2016014565) BCMA CAR 6956 SEQ ID NO. 237 (WO2016014565) BCMA CAR 6957 SEQ ID NO. 238 (WO2016014565) BCMA CAR 6958 SEQ ID NO. 239 (WO2016014565) BCMA CAR 6959 SEQ ID NO. 240 (WO2016014565) BCMA CAR 6960 SEQ ID NO. 241 (WO2016014565) BCMA CAR 6961 SEQ ID NO. 242 (WO2016014565) BCMA CAR 6962 SEQ ID NO. 243 (WO2016014565) BCMA CAR 6963 SEQ ID NO. 244 (WO2016014565) BCMA CAR 6964 SEQ ID NO. 245 (WO2016014565) BCMA CAR 6965 SEQ ID NO. 246 (WO2016014565) BCMA CAR 6966 SEQ ID NO. 247 (WO2016014565) BCMA CAR 6967 SEQ ID NO. 248 (WO2016014565) BCMA CAR 6968 SEQ ID NO. 249 (WO2016014565) BCMA CAR 6969 SEQ ID NO. 250 (WO2016014565) BCMA CAR 6970 SEQ ID NO. 251 (WO2016014565) BCMA CAR 6971 SEQ ID NO. 252 (WO2016014565) BCMA CAR 6972 SEQ ID NO. 253 (WO2016014565) BCMA CAR 6973 SEQ ID NO. 254 (WO2016014565) BCMA CAR 6974 SEQ ID NO. 267 (WO2016014565) BCMA CAR 6975 SEQ ID NO. 268 (WO2016014565) BCMA CAR 6976 SEQ ID NO. 269 (WO2016014565) BCMA CAR 6977 SEQ ID NO. 270 (WO2016014565) BCMA CAR 6978 SEQ ID NO. 1 in WO2015052538 BCMA CAR 6979 SEQ ID NO. 2 in WO2015052538 BCMA CAR 6980 SEQ ID NO. 3 in WO2015052538 BCMA CAR 6981 SEQ ID NO. 4 in WO2015052538 BCMA CAR 6982 SEQ ID NO. 5 in WO2015052538 BCMA CAR 6983 SEQ ID NO. 20 in WO2015052538 BCMA CAR 6984 SEQ ID No. 1 in US20160237139A1 BCMA CAR 6985 SEQ ID No. 2 in US20160237139A1 BCMA CAR 6986 SEQ ID No. 3 in US20160237139A1 BCMA CAR 6987 SEQ ID No. 4 in US20160237139A1 BCMA CAR 6988 SEQ ID No. 5 in US20160237139A1 BCMA CAR 6989 SEQ ID No. 6 in US20160237139A1 BCMA CAR 6990 SEQ ID NO: 9 in WO2016094304A3 BCMA CAR 6991 SEQ ID NO. 4 in WO2013154760 BCMA CAR 6992 SEQ ID NO. 5 in WO2013154760 BCMA CAR 6993 SEQ ID NO. 6 in WO2013154760 BCMA CAR 6994 SEQ ID NO. 8 in WO2013154760 BCMA CAR 6995 SEQ ID NO. 9 in WO2013154760 BCMA CAR 6996 SEQ ID NO. 10 in WO2013154760 BCMA CAR 6997 SEQ ID NO. 11 in WO2013154760 BCMA CAR 6998 SEQ ID NO. 12 in WO2013154760 BCMA CAR 6999 SEQ ID NO. 15 in WO2016014789 BCMA CAR 7000 SEQ ID NO. 16 in WO2016014789 BCMA CAR 7001 SEQ ID NO. 17 in WO2016014789 BCMA CAR 7002 SEQ ID NO. 18 in WO2016014789 BCMA CAR 7003 SEQ ID NO. 19 in WO2016014789 BCMA CAR 7004 SEQ ID NO. 20 in WO2016014789 BCMA CAR 7005 SEQ ID NO. 21 in WO2016014789 BCMA CAR 7006 SEQ ID NO. 22 in WO2016014789 BCMA CAR 7007 SEQ ID NO. 23 in WO2016014789 BCMA CAR 7008 SEQ ID NO. 24 in WO2016014789 BCMA CAR 7009 SEQ ID NO. 25 in WO2016014789 BCMA CAR 7010 SEQ ID NO. 26 in WO2016014789 BCMA CAR 7011 SEQ ID NO. 27 in WO2016014789 BCMA CAR 7012 SEQ ID NO. 28 in WO2016014789 BCMA CAR 7013 SEQ ID NO. 29 in WO2016014789 BCMA CAR 7014 SEQ ID NO. 71 in WO2016014789 BCMA CAR 7015 SEQ ID NO. 73 in WO2016014789 BCMA CAR 7016 SEQ ID NO. 125 in WO2016120216 BCMA CAR 7017 SEQ ID NO. 126 in WO2016120216 BCMA CAR 7018 SEQ ID NO. 127 in WO2016120216 BCMA CAR 7019 SEQ ID NO. 128 in WO2016120216 BCMA CAR 7020 SEQ ID NO. 129 in WO2016120216 BCMA CAR 7021 SEQ ID NO. 130 in WO2016120216 BCMA CAR 7022 SEQ ID NO. 131 in WO2016120216 BCMA CAR 7023 SEQ ID NO. 132 in WO2016120216 BCMA CAR 7024 SEQ ID NO. 133 in WO2016120216 BCMA CAR 7025 SEQ ID NO. 134 in WO2016120216 BCMA CAR 7026 SEQ ID NO. 135 in WO2016120216 BCMA CAR 7027 SEQ ID NO. 136 in WO2016120216 BCMA CAR 7028 SEQ ID NO. 137 in WO2016120216 BCMA CAR 7029 SEQ ID NO. 138 in WO2016120216 BCMA CAR 7030 SEQ ID NO. 139 in WO2016120216 BCMA CAR 7031 SEQ ID NO. 140 in WO2016120216 BCMA CAR 7032 SEQ ID NO. 141 in WO2016120216 BCMA CAR 7033 SEQ ID NO. 145 in WO2016120216 BCMA CAR 7034 SEQ ID NO. 146 in WO2016120216 BCMA CAR 7035 SEQ ID NO. 147 in WO2016120216 BCMA CAR 7036 SEQ ID NO. 148 in WO2016120216 BCMA CAR 7037 SEQ ID NO. 149 in WO2016120216 BCMA CAR 7038 SEQ ID NO. 150 in WO2016120216 BCMA NCAR 7039 SEQ ID NO: 102 in WO2016097231 BCMA NCAR 7040 SEQ ID NO: 106 in WO2016097231 BCMA NCAR 7041 SEQ ID NO: 107 in WO2016097231 BCMA NCAR 7042 SEQ ID NO: 108 in WO2016097231 BCMA NCAR 7043 SEQ ID NO: 109 in WO2016097231 BCMA NCAR 7044 SEQ ID NO: 110 in WO2016097231 BCMA NCAR 7045 SEQ ID NO: 111 in WO2016097231 BCMA NCAR 7046 SEQ ID NO: 112 in WO2016097231 BCMA NCAR 7047 SEQ ID NO: 129 in WO2016097231 BCMA NCAR 7048 SEQ ID NO: 130 in WO2016097231 BCMA NCAR 7049 SEQ ID NO: 131 in WO2016097231 BCMA NCAR 7050 SEQ ID NO: 132 in WO2016097231 BCMA NCAR 7051 SEQ ID NO: 133 in WO2016097231 BCMA NCAR 7052 SEQ ID NO: 134 in WO2016097231 BCMA NCAR 7053 SEQ ID NO: 135 in WO2016097231 BCMA NCAR 7054 SEQ ID NO: 136 in WO2016097231 BCMA NCAR 7055 SEQ ID NO: 113 in WO2016097231 BCMA NCAR 7056 SEQ ID NO: 114 in WO2016097231 BCMA NCAR 7057 SEQ ID NO: 115 in WO2016097231 BCMA NCAR 7058 SEQ ID NO: 116 in WO2016097231 BCMA NCAR 7059 SEQ ID NO: 117 in WO2016097231 BCMA NCAR 7060 SEQ ID NO: 118 in WO2016097231 BCMA NCAR 7061 SEQ ID NO: 101 in WO2016097231 BCMA NCAR 7062 SEQ ID NO: 100 in WO2016097231 BCMA NCAR 7063 SEQ ID NO: 137 in WO2016097231 BCMA NCAR 7064 SEQ ID NO: 119 in WO2016097231 BCMA NCAR 7065 SEQ ID NO: 120 in WO2016097231 BCMA NCAR 7066 SEQ ID NO: 121 in WO2016097231 BCMA NCAR 7067 SEQ ID NO: 122 in WO2016097231 BCMA NCAR 7068 SEQ ID NO: 123 in WO2016097231 BCMA NCAR 7069 SEQ ID NO: 124 in WO2016097231 BCMA NCAR 7070 SEQ ID NO: 125 in WO2016097231 BCMA NCAR 7071 SEQ ID NO: 126 in WO2016097231 BCMA NCAR 7072 SEQ ID NO: 127 in WO2016097231 BCMA NCAR 7073 SEQ ID NO: 128 in WO2016097231 BCMA NCAR 7074 SEQ ID NO: 103 in WO2016097231 BCMA NCAR 7075 SEQ ID NO: 104 in WO2016097231 BCMA NCAR 7076 SEQ ID NO: 105 in WO2016097231 BCMA NCAR 7077 SEQ ID NO: 213 in WO2016097231 CAR 7078 SEQ ID NO: 6 in US20160296562A1 CAR AND gate (CD19 AND CD33) 7079 SEQ ID NO: 2 in US20160296562A1 CD148 phosphatase CAR AND gate (CD19 AND CD5) 7080 SEQ ID NO: 43 in US20160296562A1 CAR AND gate (CD19 AND EGFRvIII) 7081 SEQ ID NO: 45 in US20160296562A1 CAR AND gate (CD19 AND GD2) 7082 SEQ ID NO: 41 in US20160296562A1 CAR AND gate (CD19 OR CD33) 7083 SEQ ID NO: 3 in US20160296562A1 CD45 phosphatase CAR AND NOT gate (CD19 AND NOT 7084 SEQ ID NO: 4 in US20160296562A1 CD33) CAR AND NOT gate (CD19 AND NOT 7085 SEQ ID NO: 5 in US20160296562A1 CD33) CAR AND NOT gate 1 7086 SEQ ID NO: 48 in US20160296562A1 CAR AND NOT gate 2 7087 SEQ ID NO: 49 in US20160296562A1 CAR AND NOT gate 3 7088 SEQ ID NO: 50 in US20160296562A1 CAR OR gate (CD19 OR CD33) 7089 SEQ ID NO: 1 in US20160296562A1 CAT19 CAR with a CD28Zeta 7090 SEQ ID NO. 12 in WO2016139487 endodomain CAT19 CAR with an OX40Zeta 7091 SEQ ID NO. 11 in WO2016139487 endodomain CAT19 chimeric gen receptor (CAR) 7092 SEQ ID NO. 10 in WO2016139487 using Campana architecture CD123 CAR 7093 SEQ ID NO. 69 in WO2016142532 CD123 CAR 7094 SEQ ID NO. 23 in WO2015140268A1 CD123 CAR 7095 SEQ ID NO. 24 in WO2015140268A1 CD123 CAR 7096 SEQ ID NO. 25 in WO2015140268A1 CD123 CAR 7097 SEQ ID NO. 26 in WO2015140268A1 CD123 CAR 7098 SEQ ID NO. 27 in WO2015140268A1 CD123 CAR 7099 SEQ ID NO. 28 in WO2015140268A1 CD123 CAR 7100 SEQ ID NO. 29 in WO2015140268A1 CD123 CAR 7101 SEQ ID NO. 30 in WO2015140268A1 CD123 CAR 7102 SEQ ID NO. 31 in WO2015140268A1 CD123 CAR 7103 SEQ ID NO. 32 in WO2015140268A1 CD123 CAR 7104 SEQ ID NO. 33 in WO2015140268A1 CD123 CAR 7105 SEQ ID NO. 34 in WO2015140268A1 CD123 CAR 7106 SEQ ID NO. 35 in WO2015140268A1 CD123 CAR 7107 SEQ ID NO. 36 in WO2015140268A1 CD123 CAR 7108 SEQ ID NO. 37 in WO2015140268A1 CD123 CAR 7109 SEQ ID NO. 38 in WO2015140268A1 CD123 CAR 7110 SEQ ID NO. 39 in WO2015140268A1 CD123 CAR 7111 SEQ ID NO. 40 in WO2015140268A1 CD123 CAR 7112 SEQ ID NO. 41 in WO2015140268A1 CD123 CAR 7113 SEQ ID NO. 42 in WO2015140268A1 CD123 CAR 7114 SEQ ID NO. 43 in WO2015140268A1 CD123 CAR 7115 SEQ ID NO. 44 in WO2015140268A1 CD123 CAR 7116 SEQ ID NO. 45 in WO2015140268A1 CD123 CAR 7117 SEQ ID NO. 46 in WO2015140268A1 CD123 CAR 7118 SEQ ID NO. 47 in WO2015140268A1 CD123 CAR 7119 SEQ ID NO. 48 in WO2015140268A1 CD123 CAR 7120 SEQ ID NO: 9 (US20140271582) CD123 CAR 7121 SEQ ID NO: 10 (US20140271582) CD123 CAR 7122 SEQ ID NO. 11 (US20140271582) CD123 CAR 7123 SEQ ID NO: 12 (US20140271582) CD123 CAR 7124 SEQ ID NO: 56 in WO2016097231 CD123 CAR 7125 SEQ ID NO. 57 in WO2016097231 CD123 CAR 7126 SEQ ID NO: 58 in WO2016097231 CD123 CAR 7127 SEQ ID NO: 59 in WO2016097231 CD123 CAR 7128 SEQ ID NO: 60 in WO2016097231 CD123 CAR 7129 SEQ ID NO: 61 in WO2016097231 CD123 CAR 7130 SEQ ID NO. 98 in WO2016028896 CD123 CAR 7131 SEQ ID NO. 99 in WO2016028896 CD123 CAR 7132 SEQ ID NO. 100 in WO2016028896 CD123 CAR 7133 SEQ ID NO. 101 in WO2016028896 CD123 CAR 7134 SEQ ID NO. 125 in WO2016028896 CD123 CAR 7135 SEQ ID NO. 126 in WO2016028896 CD123 CAR 7136 SEQ ID NO. 127 in WO2016028896 CD123 CAR 7137 SEQ ID NO. 128 in WO2016028896 CD123 CAR 7138 SEQ ID NO. 129 in WO2016028896 CD123 CAR 7139 SEQ ID NO. 130 in WO2016028896 CD123 CAR 7140 SEQ ID NO. 131 in WO2016028896 CD123 CAR 7141 SEQ ID NO. 132 in WO2016028896 CD123 CAR 7142 SEQ ID NO. 133 in WO2016028896 CD123 CAR 7143 SEQ ID NO. 134 in WO2016028896 CD123 CAR 7144 SEQ ID NO. 135 in WO2016028896 CD123 CAR 7145 SEQ ID NO. 136 in WO2016028896 CD123 CAR 7146 SEQ ID NO. 137 in WO2016028896 CD123 CAR 7147 SEQ ID NO. 138 in WO2016028896 CD123 CAR 7148 SEQ ID NO. 139 in WO2016028896 CD123 CAR 7149 SEQ ID NO. 140 in WO2016028896 CD123 CAR 7150 SEQ ID NO. 141 in WO2016028896 CD123 CAR 7151 SEQ ID NO. 142 in WO2016028896 CD123 CAR 7152 SEQ ID NO. 143 in WO2016028896 CD123 CAR 7153 SEQ ID NO. 144 in WO2016028896 CD123 CAR 7154 SEQ ID NO. 145 in WO2016028896 CD123 CAR 7155 SEQ ID NO. 146 in WO2016028896 CD123 CAR 7156 SEQ ID NO. 147 in WO2016028896 CD123 CAR 7157 SEQ ID NO. 148 in WO2016028896 CD123 CAR 7158 SEQ ID NO. 149 in WO2016028896 CD123 CAR 7159 SEQ ID NO. 150 in WO2016028896 CD123 CAR 7160 SEQ ID NO. 151 in WO2016028896 CD123 CAR 7161 SEQ ID NO. 152 in WO2016028896 CD123 CAR 7162 SEQ ID NO. 153 in WO2016028896 CD123 CAR 7163 SEQ ID NO. 154 in WO2016028896 CD123 CAR 7164 SEQ ID NO. 155 in WO2016028896 CD123 CAR 7165 SEQ ID NO. 156 in WO2016028896 CD123 CAR 7166 SEQ ID NO. 31 in WO2016120220 CD123 CAR 7167 SEQ ID NO. 32 in WO2016120220 CD123 CAR 7168 SEQ ID NO. 33 in WO2016120220 CD123 CAR 7169 SEQ ID NO. 34 in WO2016120220 CD123 CAR 7170 SEQ ID NO. 35 in WO2016120220 CD123 CAR 7171 SEQ ID NO. 36 in WO2016120220 CD123 CAR 7172 SEQ ID NO. 37 in WO2016120220 CD123 CAR 7173 SEQ ID NO. 38 in WO2016120220 CD123 CAR 7174 SEQ ID NO. 39 in WO2016120220 CD123 CAR 7175 SEQ ID NO. 40 in WO2016120220 CD123 CAR 7176 SEQ ID NO. 41 in WO2016120220 CD123 CAR 7177 SEQ ID NO. 42 in WO2016120220 CD123 CAR 7178 SEQ ID NO. 43 in WO2016120220 CD123 CAR 7179 SEQ ID NO. 44 in WO2016120220 CD123 CAR 7180 SEQ ID NO. 45 in WO2016120220 CD123 CAR 7181 SEQ ID NO. 46 in WO2016120220 CD123 CAR 7182 SEQ ID NO. 47 in WO2016120220 CD123 CAR 7183 SEQ ID NO. 48 in WO2016120220 CD123 CAR 7184 SEQ ID NO. 49 in WO2016120220 CD123 CAR 7185 SEQ ID NO. 50 in WO2016120220 CD123 CAR 7186 SEQ ID NO. 51 in WO2016120220 CD123 CAR 7187 SEQ ID NO. 52 in WO2016120220 CD123 CAR 7188 SEQ ID NO. 53 in WO2016120220 CD123 CAR 7189 SEQ ID NO. 54 in WO2016120220 CD123 CAR 7190 SEQ ID NO. 55 in WO2016120220 CD123 CAR 7191 SEQ ID NO. 56 in WO2016120220 CD123 CAR 7192 SEQ ID NO. 57 in WO2016120220 CD123 CAR 7193 SEQ ID NO. 58 in WO2016120220 CD123 CAR 7194 SEQ ID NO. 59 in WO2016120220 CD123 CAR 7195 SEQ ID NO. 60 in WO2016120220 CD123 CAR 7196 SEQ ID NO. 61 in WO2016120220 CD123 CAR 7197 SEQ ID NO. 62 in WO2016120220 CD123 CAR 7198 SEQ ID NO. 63 in WO2016120220 CD123 CAR 7199 SEQ ID NO. 64 in WO2016120220 CD123 CAR 7200 SEQ ID NO. 65 in WO2016120220 CD123 CAR 7201 SEQ ID NO. 66 in WO2016120220 CD123 CAR 7202 SEQ ID NO. 67 in WO2016120220 CD123 CAR 7203 SEQ ID NO. 68 in WO2016120220 CD123 CAR 7204 SEQ ID NO. 69 in WO2016120220 CD123 CAR 7205 SEQ ID NO. 70 in WO2016120220 CD123 CAR 7206 SEQ ID NO. 71 in WO2016120220 CD123 CAR 7207 SEQ ID NO. 72 in WO2016120220 CD123 CAR 7208 SEQ ID NO. 73 in WO2016120220 CD123 CAR 7209 SEQ ID NO. 74 in WO2016120220 CD123 CAR 7210 SEQ ID NO. 75 in WO2016120220 CD123 CAR 7211 SEQ ID NO. 76 in WO2016120220 CD123 CAR 7212 SEQ ID NO. 77 in WO2016120220 CD123 CAR 7213 SEQ ID NO. 78 in WO2016120220 CD123 CAR 7214 SEQ ID NO. 79 in WO2016120220 CD123 CAR 7215 SEQ ID NO. 80 in WO2016120220 CD123 CAR 7216 SEQ ID NO. 81 in WO2016120220 CD123 CAR 7217 SEQ ID NO. 82 in WO2016120220 CD123 CAR 7218 SEQ ID NO. 83 in WO2016120220 CD123 CAR 7219 SEQ ID NO. 84 in WO2016120220 CD123 CAR 7220 SEQ ID NO. 85 in WO2016120220 CD123 CAR 7221 SEQ ID NO. 86 in WO2016120220 CD123 CAR 7222 SEQ ID NO. 87 in WO2016120220 CD123 CAR 7223 SEQ ID NO. 88 in WO2016120220 CD123 CAR 7224 SEQ ID NO. 89 in WO2016120220 CD123 CAR 7225 SEQ ID NO. 90 in WO2016120220 CD123 CAR 7226 SEQ ID NO. 91 in WO2016120220 CD123 CAR 7227 SEQ ID NO. 92 in WO2016120220 CD123 CAR 7228 SEQ ID NO. 93 in WO2016120220 CD123 CAR 7229 SEQ ID NO. 94 in WO2016120220 CD123 CAR 7230 SEQ ID NO. 95 in WO2016120220 CD123 CAR 7231 SEQ ID NO. 96 in WO2016120220 CD123 CAR 7232 SEQ ID NO. 97 in WO2016120220 CD123 CAR 7233 SEQ ID NO. 98 in WO2016120220 CD123 CAR 7234 SEQ ID NO. 99 in WO2016120220 CD123 CAR 7235 SEQ ID NO. 100 in WO2016120220 CD123 CAR 7236 SEQ ID NO. 101 in WO2016120220 CD123 CAR 7237 SEQ ID NO. 102 in WO2016120220 CD123 CAR 7238 SEQ ID NO. 103 in WO2016120220 CD123 CAR 7239 SEQ ID NO. 104 in WO2016120220 CD123 CAR 7240 SEQ ID NO. 105 in WO2016120220 CD123 CAR 7241 SEQ ID NO. 106 in WO2016120220 CD123 CAR 7242 SEQ ID NO. 107 in WO2016120220 CD123 CAR 7243 SEQ ID NO. 108 in WO2016120220 CD123 CAR 7244 SEQ ID NO. 109 in WO2016120220 CD123 CAR 7245 SEQ ID NO. 110 in WO2016120220 CD123 CAR 7246 SEQ ID NO. 111 in WO2016120220 CD123 CAR 7247 SEQ ID NO. 112 in WO2016120220 CD123 CAR 7248 SEQ ID NO. 113 in WO2016120220 CD123 CAR 7249 SEQ ID NO. 114 in WO2016120220 CD123 CAR 7250 SEQ ID NO. 115 in WO2016120220 CD123 CAR 7251 SEQ ID NO. 116 in WO2016120220 CD123 CAR 7252 SEQ ID NO. 117 in WO2016120220 CD123 CAR 7253 SEQ ID NO. 118 in WO2016120220 CD123 CAR 7254 SEQ ID NO. 119 in WO2016120220 CD123 CAR 7255 SEQ ID NO. 120 in WO2016120220 CD123 CAR 7256 SEQ ID NO. 121 in WO2016120220 CD123 CAR 7257 SEQ ID NO. 122 in WO2016120220 CD123 CAR 7258 SEQ ID NO. 123 in WO2016120220 CD123 CAR 7259 SEQ ID NO. 124 in WO2016120220 CD123 CAR 7260 SEQ ID NO. 125 in WO2016120220 CD123 CAR 7261 SEQ ID NO. 126 in WO2016120220 CD123 CAR 7262 SEQ ID NO. 127 in WO2016120220 CD123 CAR 7263 SEQ ID NO. 128 in WO2016120220 CD123 CAR 7264 SEQ ID NO. 129 in WO2016120220 CD123 CAR 7265 SEQ ID NO. 130 in WO2016120220 CD123 CAR 7266 SEQ ID NO. 131 in WO2016120220 CD123 CAR 7267 SEQ ID NO. 132 in WO2016120220 CD123 CAR 7268 SEQ ID NO. 133 in WO2016120220 CD123 CAR 7269 SEQ ID NO. 134 in WO2016120220 CD123 CAR 7270 SEQ ID NO. 135 in WO2016120220 CD123 CAR 7271 SEQ ID NO. 136 in WO2016120220 CD123 CAR 7272 SEQ ID NO. 137 in WO2016120220 CD123 CAR 7273 SEQ ID NO. 138 in WO2016120220 CD123 CAR 7274 SEQ ID NO. 139 in WO2016120220 CD123 CAR 7275 SEQ ID NO. 140 in WO2016120220 CD123 CAR 7276 SEQ ID NO. 141 in WO2016120220 CD123 CAR 7277 SEQ ID NO. 142 in WO2016120220 CD123 CAR 7278 SEQ ID NO. 143 in WO2016120220 CD123 CAR 7279 SEQ ID NO. 144 in WO2016120220 CD123 CAR 7280 SEQ ID NO. 145 in WO2016120220 CD123 CAR 7281 SEQ ID NO. 146 in WO2016120220 CD123 CAR 7282 SEQ ID NO. 147 in WO2016120220 CD123 CAR 7283 SEQ ID NO. 148 in WO2016120220 CD123 CAR 7284 SEQ ID NO. 149 in WO2016120220 CD123 CAR 7285 SEQ ID NO. 150 in WO2016120220 CD123 CAR 7286 SEQ ID NO. 151 in WO2016120220 CD123 CAR 7287 SEQ ID NO. 152 in WO2016120220 CD123 CAR 7288 SEQ ID NO. 153 in WO2016120220 CD123 CAR 7289 SEQ ID NO. 154 in WO2016120220 CD123 CAR 7290 SEQ ID NO. 155 in WO2016120220 CD123 CAR 7291 SEQ ID NO. 156 in WO2016120220 CD123 CAR 7292 SEQ ID NO. 157 in WO2016120220 CD123 CAR 7293 SEQ ID NO. 158 in WO2016120220 CD123 CAR 7294 SEQ ID NO. 159 in WO2016120220 CD123 CAR 7295 SEQ ID NO. 160 in WO2016120220 CD123 CAR 7296 SEQ ID NO. 172 in WO2016120220 CD123 CAR 7297 SEQ ID NO. 173 in WO2016120220 CD123 CAR 7298 SEQ ID NO. 174 in WO2016120220 CD123 CAR 7299 SEQ ID NO. 175 in WO2016120220 CD123 CAR 7300 SEQ ID NO. 176 in WO2016120220 CD123 CAR 7301 SEQ ID NO. 177 in WO2016120220 CD123 CAR 7302 SEQ ID NO. 178 in WO2016120220 CD123 CAR 7303 SEQ ID NO. 179 in WO2016120220 CD123 CAR 7304 SEQ ID NO. 180 in WO2016120220 CD123 CAR 7305 SEQ ID NO. 181 in WO2016120220 CD123 CAR 7306 SEQ ID NO. 182 in WO2016120220 CD123 CAR 7307 SEQ ID NO. 183 in WO2016120220 CD123 CAR 7308 SEQ ID NO. 184 in WO2016120220 CD123 CAR 7309 SEQ ID NO. 185 in WO2016120220 CD123 CAR 7310 SEQ ID NO. 186 in WO20I6120220 CD123 CAR 7311 SEQ ID NO. 187 in WO2016120220 CD123 CAR 7312 SEQ ID NO. 188 in WO2016120220 CD123 CAR 7313 SEQ ID NO. 189 in WO2016120220 CD123 CAR 7314 SEQ ID NO. 190 in WO2016120220 CD123 CAR 7315 SEQ ID NO. 191 in WO2016120220 CD123 CAR 7316 SEQ ID NO. 192 in WO2016120220 CD123 CAR 7317 SEQ ID NO. 193 in WO2016120220 CD123 CAR 7318 SEQ ID NO. 194 in WO2016120220 CD123 CAR 7319 SEQ ID NO. 195 in WO2016120220 CD123 CAR 7320 SEQ ID NO. 196 in WO2016120220 CD123 CAR 7321 SEQ ID NO. 197 in WO2016120220 CD123 CAR 7322 SEQ ID NO. 1 in WO2016120216 CD123 CAR 7323 SEQ ID NO. 2 in WO2016120216 CD123 CAR 7324 SEQ ID NO. 3 in WO2016120216 CD123 CAR 7325 SEQ ID NO. 4 in WO2016120216 CD123 CAR 7326 SEQ ID NO. 5 in WO2016120216 CD123 CAR 7327 SEQ ID NO. 6 in WO2016120216 CD123 CAR 7328 SEQ ID NO. 7 in WO2016120216 CD123 CAR 7329 SEQ ID NO. 8 in WO2016120216 CD123 CAR 7330 SEQ ID NO. 9 in WO2016120216 CD123 CAR 7331 SEQ ID NO. 10 in WO2016120216 CD123 CAR 7332 SEQ ID NO. 142 in WO2016120216 CD19/IL13 Bispecific CAR 7333 SEQ ID NO: 10 in US20160340649A1 CD19 CAR 7334 SEQ ID NO: 12 in U.S. Pat. No. 9,499,629B2 CD19 CAR 7335 SEQ ID NO: 24 in US20160333108A1 CD19 CAR 7336 SEQ ID NO. 25 in US20160333108A1 CD19 CAR 7337 SEQ ID NO: 26 in US20160333108A1 CD19 CAR 7338 SEQ ID NO: 27 in US20160333108A1 CD19 CAR 7339 SEQ ID NO: 1 in EP2997134A4 CD19 CAR 7340 SEQ ID NO: 19 in EP3071687A1 CD19 CAR 7341 SEQ ID NO: 20 in EP3071687A1 CD19 CAR 7342 SEQ ID NO: 181 in WO2016168773A3 CD19 CAR 7343 SEQ ID NO: 2 in WO2015157399A9 CD19 CAR 7344 SEQ ID NO: 56 in WO2016174409A1 CD19 CAR 7345 SEQ ID NO: 62 in WO2016174409A1 CD19 CAR 7346 SEQ ID NO: 145 in WO2016179319A1 CD19 CAR 7347 SEQ ID NO: 293 in US20160311907A1 CD19 CAR 7348 SEQ ID NO: 294 in US20160311907A1 CD19 CAR 7349 SEQ ID NO: 295 in US20160311907A1 CD19 CAR 7350 SEQ ID NO: 296 in US20160311907A1 CD19 CAR 7351 SEQ ID NO: 297 in US20160311907A1 CD19 CAR 7352 SEQ ID NO: 298 in US20160311907A1 CD19 CAR 7353 SEQ ID NO. 73 in WO2013176915A1 CD19 CAR 7354 SEQ ID NO. 73 in WO2013176916A1 CD19 CAR 7355 SEQ ID NO. 73 in US20130315884A1 CD19 CAR 7356 SEQ ID NO. 73 in US20140134142A1 CD19 CAR 7357 SEQ ID NO. 73 in US20150017136A1 CD19 CAR 7358 SEQ ID NO. 73 in US20150203817A1 CD19 CAR 7359 SEQ ID NO. 73 in US20160120905A1 CD19 CAR 7360 SEQ ID NO. 73 in US20160120906A1 CD19 CAR 7361 SEQ ID NO. 8 in WO2015124715 CD19 CAR 7362 SEQ ID NO. 5 in WO2015124715 CD19 CAR 7363 SEQ ID NO. 73 in WO2014184744 CD19 CAR 7364 SEQ ID NO. 73 in WO2014184741 CD19 CAR 7365 SEQ ID NO. 14 in US20160145337A1 CD19 CAR 7366 SEQ ID NO. 15 in US20160145337A1 CD19 CAR 7367 SEQ ID NO. 14 in WO2014184143 CD19 CAR 7368 SEQ ID NO. 15 in WO2014184143 CD19 CAR 7369 SEQ ID NO. 15 in WO2015075175 CD19 CAR 7370 SEQ ID NO. 16 in WO2015075175 CD19 CAR 7371 SEQ ID NO. 16 in US20160145337A1 CD19 CAR 7372 SEQ ID NO. 16 in WO2014184143 CD19 CAR 7373 SEQ ID NO 12 in WO2012079000 CD19 CAR 7374 SEQ ID NO. 31 in WO2016164580 CD19 CAR 7375 SEQ ID NO. 32 in WO2016164580 CD19 CAR 7376 SEQ ID NO. 33 in WO2016164580 CD19 CAR 7377 SEQ ID NO. 34 in WO2016164580 CD19 CAR 7378 SEQ ID NO. 35 in WO2016164580 CD19 CAR 7379 SEQ ID NO. 36 in WO2016164580 CD19 CAR 7380 SEQ ID NO. 37 in WO2016164580 CD19 CAR 7381 SEQ ID NO. 38 in WO2016164580 CD19 CAR 7382 SEQ ID NO. 39 in WO2016164580 CD19 CAR 7383 SEQ ID NO. 40 in WO2016164580 CD19 CAR 7384 SEQ ID NO. 41 in WO2016164580 CD19 CAR 7385 SEQ ID NO. 42 in WO2016164580 CD19 CAR 7386 SEQ ID NO. 58 in WO2016164580 CD19 CAR 7387 SEQ ID NO: 14 in US20160296563A1 CD19 CAR 7388 SEQ ID NO: 15 in US20160296563A1 CD19 CAR 7389 SEQ ID NO. 31 in WO2015157252 CD19 CAR 7390 SEQ ID NO. 32 in WO2015157252 CD19 CAR 7391 SEQ ID NO. 33 in WO2015157252 CD19 CAR 7392 SEQ ID NO. 34 in WO2015157252 CD19 CAR 7393 SEQ ID NO. 35 in WO2015157252 CD19 CAR 7394 SEQ ID NO. 36 in WO2015157252 CD19 CAR 7395 SEQ ID NO. 37 in WO2015157252 CD19 CAR 7396 SEQ ID NO. 38 in WO2015157252 CD19 CAR 7397 SEQ ID NO. 39 in WO2015157252 CD19 CAR 7398 SEQ ID NO. 40 in WO2015157252 CD19 CAR 7399 SEQ ID NO. 41 in WO2015157252 CD19 CAR 7400 SEQ ID NO. 42 in WO2015157252 CD19 CAR 7401 SEQ ID NO. 14 in WO2016139487 CD19 CAR 7402 SEQ ID NO. 15 in WO2016139487 CD19 CAR 7403 SEQ ID NO: 53 in US20160250258A1 CD19 CAR 7404 SEQ ID NO: 54 in US20160250258A1 CD19 CAR 7405 SEQ ID NO: 55 in US20160250258A1 CD19 CAR 7406 SEQ ID NO: 56 in US20160250258A1 CD19 CAR 7407 SEQ ID NO: 57 in US20160250258A1 CD19 CAR 7408 SEQ ID NO: 58 in US20160250258A1 CD19 CAR 7409 SEQ ID NO. 1 in WO2015187528 CD19 CAR 7410 SEQ ID NO. 2 in WO2015187528 CD19 CAR 7411 SEQ ID NO. 3 in WO2015187528 CD19 CAR 7412 SEQ ID NO. 4 in WO2015187528 CD19 CAR 7413 SEQ ID NO. 5 in WO2015187528 CD19 CAR 7414 SEQ ID NO. 6 in WO2015187528 CD19 CAR 7415 SEQ ID NO. 7 in WO2015187528 CD19 CAR 7416 SEQ ID NO. 8 in WO2015187528 CD19 CAR 7417 SEQ ID NO. 9 in WO2015187528 CD19 CAR 7418 SEQ ID NO. 10 in WO2015187528 CD19 CAR 7419 SEQ ID NO. 11 in WO2015187528 CD19 CAR 7420 SEQ ID NO. 12 in WO2015187528 CD19 CAR 7421 SEQ ID NO. 13 in WO2015187528 CD19 CAR 7422 SEQ ID. NO. 31 in WO2015157252 CD19 CAR 7423 SEQ ID. NO. 32 in WO2015157252 CD19 CAR 7424 SEQ ID. NO. 33 in WO2015157252 CD19 CAR 7425 SEQ ID. NO. 34 in WO2015157252 CD19 CAR 7426 SEQ ID. NO. 35 in WO2015157252 CD19 CAR 7427 SEQ ID. NO. 36 in WO2015157252 CD19 CAR 7428 SEQ ID. NO. 37 in WO2015157252 CD19 CAR 7429 SEQ ID. NO. 38 in WO2015157252 CD19 CAR 7430 SEQ ID. NO. 39 in WO2015157252 CD19 CAR 7431 SEQ ID. NO. 40 in WO2015157252 CD19 CAR 7432 SEQ ID. NO. 41 in WO2015157252 CD19 CAR 7433 SEQ ID. NO. 42 in WO2015157252 CD19 CAR 7434 SEQ ID. NO. 58 in WO2015157252 CD19 CAR 7435 SEQ ID NO. 31 in WO2014153270 CD19 CAR 7436 SEQ ID NO. 32 in WO2014153270 CD19 CAR 7437 SEQ ID NO. 33 in WO2014153270 CD19 CAR 7438 SEQ ID NO. 34 in WO2014153270 CD19 CAR 7439 SEQ ID NO. 35 in WO2014153270 CD19 CAR 7440 SEQ ID NO. 36 in WO2014153270 CD19 CAR 7441 SEQ ID NO. 37 in WO2014153270 CD19 CAR 7442 SEQ ID NO. 38 in WO2014153270 CD19 CAR 7443 SEQ ID NO. 39 in WO2014153270 CD19 CAR 7444 SEQ ID NO. 40 in WO2014153270 CD19 CAR 7445 SEQ ID NO. 41 in WO2014153270 CD19 CAR 7446 SEQ ID NO. 42 in WO2014153270 CD19 CAR 7447 in WO2016134284 (no SEQ ID NO) CD19 CAR (Third generation) 7448 SEQ ID NO. 13 in WO2016139487 CD19 or CD33 CAR (a CAR OR gate 7449 SEQ ID NO. 1 in WO2015075468 which recognizes CD19 OR CD33) CD19CD20 CAR Bispecific CAR 7450 SEQ ID NO: 1308 in WO2016164731A2 CD19CD20 CAR Bispecific CAR 7451 SEQ ID NO: 2 in U.S. Pat. No. 9,447,194B2 CD19CD20 CAR Bispecific CAR 7452 SEQ ID NO: 8 in U.S. Pat. No. 9,447,194B2 CD19CD20 CAR Bispecific CAR 7453 SEQ ID NO: 11 in U.S. Pat. No. 9,447,194B2 CD2 CAR 7454 SEQ ID NO. 10 in WO2016138491 CD2 CAR 7455 SEQ ID NO. 11 in WO2016138491 CD20 CAR 7456 SEQ ID NO: 25 in WO2015157399A9 CD20 NCAR 7457 SEQ ID NO: 177 in WO2016097231 CD20 NCAR 7458 SEQ ID NO: 181 in WO2016097231 CD20 NCAR 7459 SEQ ID NO: 182 in WO2016097231 CD20 NCAR 7460 SEQ ID NO: 183 in WO2016097231 CD20 NCAR 7461 SEQ ID NO: 184 in WO2016097231 CD20 NCAR 7462 SEQ ID NO: 185 in WO2016097231 CD20 NCAR 7463 SEQ ID NO: 186 in WO2016097231 CD20 NCAR 7464 SEQ ID NO: 187 in WO2016097231 CD20 NCAR 7465 SEQ ID NO: 205 in WO2016097231 CD20 NCAR 7466 SEQ ID NO: 206 in WO2016097231 CD20 NCAR 7467 SEQ ID NO: 207 in WO2016097231 CD20 NCAR 7468 SEQ ID NO: 208 in WO2016097231 CD20 NCAR 7469 SEQ ID NO: 209 in WO2016097231 CD20 NCAR 7470 SEQ ID NO: 210 in WO2016097231 CD20 NCAR 7471 SEQ ID NO: 211 in WO2016097231 CD20 NCAR 7472 SEQ ID NO: 188 in WO2016097231 CD20 NCAR 7473 SEQ ID NO: 189 in WO2016097231 CD20 NCAR 7474 SEQ ID NO: 190 in WO2016097231 CD20 NCAR 7475 SEQ ID NO: 191 in WO2016097231 CD20 NCAR 7476 SEQ ID NO: 192 in WO2016097231 CD20 NCAR 7477 SEQ ID NO: 193 in WO2016097231 CD20 NCAR 7478 SEQ ID NO: 176 in WO2016097231 CD20 NCAR 7479 SEQ ID NO: 212 in WO2016097231 CD20 NCAR 7480 SEQ ID NO: 194 in WO2016097231 CD20 NCAR 7481 SEQ ID NO: 195 in WO2016097231 CD20 NCAR 7482 SEQ ID NO: 196 in WO2016097231 CD20 NCAR 7483 SEQ ID NO: 197 in WO2016097231 CD20 NCAR 7484 SEQ ID NO: 198 in WO2016097231 CD20 NCAR 7485 SEQ ID NO: 199 in WO2016097231 CD20 NCAR 7486 SEQ ID NO: 200 in WO2016097231 CD20 NCAR 7487 SEQ ID NO. 201 in WO2016097231 CD20 NCAR 7488 SEQ ID NO: 202 in WO2016097231 CD20 NCAR 7489 SEQ ID NO: 203 in WO2016097231 CD20 NCAR 7490 SEQ ID NO: 178 in WO2016097231 CD20 NCAR 7491 SEQ ID NO: 179 in WO2016097231 CD20 NCAR 7492 SEQ ID NO: 180 in WO2016097231 CD22 CAR 7493 SEQ ID NO: 380 in WO2016164731A2 CD22 CAR 7494 SEQ ID NO: 204 in WO2016164731A2 CD22 CAR 7495 SEQ ID NO: 260 in WO2016164731A2 CD22 CAR 7496 SEQ ID NO: 266 in WO2016164731A2 CD22 CAR 7497 SEQ ID NO: 272 in WO2016164731A2 CD22 CAR 7498 SEQ ID NO. 278 in WO2016164731A2 CD22 CAR 7499 SEQ ID NO: 284 in WO2016164731A2 CD22 CAR 7500 SEQ ID NO: 290 in WO2016164731A2 CD22 CAR 7501 SEQ ID NO: 296 in WO2016164731A2 CD22 CAR 7502 SEQ ID NO: 302 in WO2016164731A2 CD22 CAR 7503 SEQ ID NO: 308 in WO2016164731A2 CD22 CAR 7504 SEQ ID NO: 314 in WO2016164731A2 CD22 CAR 7505 SEQ ID NO: 213 in WO2016164731A2 CD22 CAR 7506 SEQ ID NO: 320 in WO2016164731A2 CD22 CAR 7507 SEQ ID NO: 326 in WO2016164731A2 CD22 CAR 7508 SEQ ID NO: 332 in WO2016164731A2 CD22 CAR 7509 SEQ ID NO: 338 in WO2016164731A2 CD22 CAR 7510 SEQ ID NO: 347 in WO2016164731A2 CD22 CAR 7511 SEQ ID NO: 350 in WO2016164731A2 CD22 CAR 7512 SEQ ID NO: 356 in WO2016164731A2 CD22 CAR 7513 SEQ ID NO: 362 in WO2016164731A2 CD22 CAR 7514 SEQ ID NO: 368 in WO2016164731A2 CD22 CAR 7515 SEQ ID NO: 374 in WO2016164731A2 CD22 CAR 7516 SEQ ID NO: 219 in WO2016164731A2 CD22 CAR 7517 SEQ ID NO: 386 in WO2016164731A2 CD22 CAR 7518 SEQ ID NO: 392 in WO2016164731A2 CD22 CAR 7519 SEQ ID NO: 398 in WO2016164731A2 CD22 CAR 7520 SEQ ID NO: 404 in WO2016164731A2 CD22 CAR 7521 SEQ ID NO: 410 in WO2016164731A2 CD22 CAR 7522 SEQ ID NO: 416 in WO2016164731A2 CD22 CAR 7523 SEQ ID NO: 421 in WO2016164731A2 CD22 CAR 7524 SEQ ID NO: 427 in WO2016164731A2 CD22 CAR 7525 SEQ ID NO: 225 in WO2016164731A2 CD22 CAR 7526 SEQ ID NO: 230 in WO2016164731A2 CD22 CAR 7527 SEQ ID NO: 1109 in WO2016164731A2 CD22 CAR 7528 SEQ ID NO: 236 in WO2016164731A2 CD22 CAR 7529 SEQ ID NO: 242 in WO2016164731A2 CD22 CAR 7530 SEQ ID NO: 248 in WO2016164731A2 CD22 CAR 7531 SEQ ID NO: 254 in WO2016164731A2 CD22 CAR 7532 SEQ ID NO. 15 in WO2013059593 CD22 CAR 7533 SEQ ID NO. 16 in WO2013059593 CD22 CAR 7534 SEQ ID NO. 17 in WO2013059593 CD22 CAR 7535 SEQ ID NO. 18 in WO2013059593 CD22 CAR 7536 SEQ ID NO. 19 in WO2013059593 CD22 CAR 7537 SEQ ID NO. 20 in WO2013059593 CD22 CAR 7538 SEQ ID NO. 32 in WO2013059593 CD22 CAR 7539 SEQ ID NO. 22 in US20150299317 CD22 CAR 7540 SEQ ID NO. 23 in US20150299317 CD22 CAR 7541 SEQ ID NO. 24 in US20150299317 CD22CD19 Bispecific CAR 7542 SEQ ID NO. 29 in WO2016149578 CD22CD19 Bispecific CAR 7543 SEQ ID NO. 30 in WO2016149578 CD22CD19 Bispecific CAR 7544 SEQ ID NO: 1304 in WO2016164731A2 CD276 CAR 7545 SEQ ID NO. 39 in US20160053017 CD276 CAR 7546 SEQ ID NO. 40 in US20160053017 CD276 CAR 7547 SEQ ID NO. 41 in US20160053017 CD276 CAR 7548 SEQ ID NO. 42 in US20160053017 CD276 CAR 7549 SEQ ID NO. 43 in US20160053017 CD276 CAR 7550 SEQ ID NO. 44 in US20160053017 CD276 CAR 7551 SEQ ID NO. 45 in US20160053017 CD276 CAR 7552 SEQ ID NO. 46 in US20160053017 CD276 CAR 7553 SEQ ID NO. 47 in US20160053017 CD276 CAR 7554 SEQ ID NO. 122 in US20160053017 CD276 CAR 7555 SEQ ID NO. 123 in US20160053017 CD276 CAR 7556 SEQ ID NO. 124 in US20160053017 CD276 CAR 7557 SEQ ID NO. 125 in US20160053017 CD276 CAR 7558 SEQ ID NO. 126 in US20160053017 CD276 CAR 7559 SEQ ID NO. 127 in US20160053017 CD276 CAR 7560 SEQ ID NO. 128 in US20160053017 CD276 CAR 7561 SEQ ID NO. 129 in US20160053017 CD276 CAR 7562 SEQ ID NO. 130 in US20160053017 CD3 CAR 7563 SEQ ID NO. 12 in WO2016138491 CD30 CAR 7564 SEQ ID NO. 20 in WO2016008973A1 CD30 CAR 7565 SEQ ID NO 1 in WO2016116035A1 CD30 CAR 7566 in WO2016134284 (no SEQ ID NO) CD30 CAR 7567 SEQ ID NO. 2 in WO2016008973A1 CD33 CAR 7568 SEQ ID NO. 48 in WO2016014576 CD33 CAR 7569 SEQ ID NO. 49 in WO2016014576 CD33 CAR 7570 SEQ ID NO. 50 in WO2016014576 CD33 CAR 7571 SEQ ID NO. 51 in WO2016014576 CD33 CAR 7572 SEQ ID NO. 52 in WO2016014576 CD33 CAR 7573 SEQ ID NO. 53 in WO2016014576 CD33 CAR 7574 SEQ ID NO. 54 in WO2016014576 CD33 CAR 7575 SEQ ID NO. 55 in WO2016014576 CD33 CAR 7576 SEQ ID NO. 83 in WO2016014576 CD33 CAR 7577 SEQ ID NO. 19 in WO2015150526A2 CD33 CAR 7578 SEQ ID NO. 20 in WO2015150526A2 CD33 CAR 7579 SEQ ID NO. 21 in WO2015150526A2 CD33 CAR 7580 SEQ ID NO. 22 in WO2015150526A2 CD33 CAR 7581 SEQ ID NO. 23 in WO2015150526A2 CD33 CAR 7582 SEQ ID NO. 24 in WO2015150526A2 CD33 CAR 7583 SEQ ID NO. 25 in WO2015150526A2 CD33 CAR 7584 SEQ ID NO. 26 in WO2015150526A2 CD33 CAR 7585 SEQ ID NO. 27 in WO2015150526A2 CD33 CAR 7586 SEQ ID NO. 28 in WO2015150526A2 CD33 CAR 7587 SEQ ID NO. 29 in WO2015150526A2 CD33 CAR 7588 SEQ ID NO. 30 in WO2015150526A2 CD33 CAR 7589 SEQ ID NO. 31 in WO2015150526A2 CD33 CAR 7590 SEQ ID NO. 32 in WO2015150526A2 CD33 CAR 7591 SEQ ID NO. 33 in WO2015150526A2 CD33 CAR 7592 SEQ ID NO. 34 in WO2015150526A2 CD33 CAR 7593 SEQ ID NO. 35 in WO2015150526A2 CD33 CAR 7594 SEQ ID NO. 36 in WO2015150526A2 CD33 CAR 7595 SEQ ID NO. 37 in WO2015150526A2 CD33 CAR 7596 SEQ ID NO. 38 in WO2015150526A2 CD33 CAR 7597 SEQ ID NO. 39 in WO2015150526A2 CD33 CAR 7598 SEQ ID NO. 40 in WO2015150526A2 CD33 CAR 7599 SEQ ID NO. 41 in WO2015150526A2 CD33 CAR 7600 SEQ ID NO. 42 in WO2015150526A2 CD33 CAR 7601 SEQ ID NO. 48 in WO2015150526A2 CD33 CAR 7602 SEQ ID NO. 49 in WO2015150526A2 CD33 CAR 7603 SEQ ID NO. 50 in WO2015150526A2 CD33 CAR 7604 SEQ ID NO. 51 in WO2015150526A2 CD33 CAR 7605 SEQ ID NO. 52 in WO2015150526A2 CD33 CAR 7606 SEQ ID NO. 53 in WO2015150526A2 CD33 CAR 7607 SEQ ID NO. 54 in WO2015150526A2 CD33 CAR 7608 SEQ ID NO. 55 in WO2015150526A2 CD33 CAR 7609 SEQ ID NO. 56 in WO2015150526A2 CD33 CAR 7610 SEQ ID NO. 57 in WO2015150526A2 CD33 CAR 7611 SEQ ID NO. 58 in WO2015150526A2 CD33 CAR 7612 SEQ ID NO. 59 in WO2015150526A2 CD33 CAR 7613 SEQ ID NO. 60 in WO2015150526A2 CD33 CAR 7614 SEQ ID NO. 61 in WO2015150526A2 CD33 CAR 7615 SEQ ID NO. 62 in WO2015150526A2 CD33 CAR 7616 SEQ ID NO. 63 in WO2015150526A2 CD33 CAR 7617 SEQ ID NO. 64 in WO2015150526A2 CD33 CAR 7618 SEQ ID NO. 65 in WO2015150526A2 CD33 CAR 7619 SEQ ID NO. 66 in WO2015150526A2 CD33 CAR 7620 SEQ ID NO. 67 in WO2015150526A2 CD33 CAR 7621 SEQ ID NO. 68 in WO2015150526A2 CD33 CAR 7622 SEQ ID NO. 69 in WO2015150526A2 CD33 CAR 7623 SEQ ID NO. 70 in WO2015150526A2 CD33 CAR 7624 SEQ ID NO. 71 in WO2015150526A2 CD38 CAR 7625 SEQ ID NO: 70 in WO2016097231 CD38 CAR 7626 SEQ ID NO: 71 in WO2016097231 CD38 CAR 7627 SEQ ID NO: 72 in WO2016097231 CD38 CAR 7628 SEQ ID NO: 64 in WO2016097231 CD38 CAR 7629 SEQ ID NO: 65 in WO2016097231 CD38 CAR 7630 SEQ ID NO: 66 in WO2016097231 CD38 CAR 7631 SEQ ID NO: 67 in WO2016097231 CD38 CAR 7632 SEQ ID NO: 68 in WO2016097231 CD38 CAR 7633 SEQ ID NO: 69 in WO2016097231 CD38 CAR 7634 SEQ ID No. 35 in WO2015121454 CD38 CAR 7635 SEQ ID No. 36 in WO2015121454 CD38 CAR 7636 SEQ ID No. 37 in WO2015121454 CD4 CAR 7637 SEQ ID NO. 13 in WO2016138491 CD4 CAR 7638 SEQ ID NO. 14 in WO2016138491 CD410 CAR 7639 SEQ ID NO: 7 in EP3074419A2 CD435 CAR 7640 SEQ ID NO: 5 in EP3074419A2 CD44 CAR 7641 SEQ ID NO. 21 in WO2016042461A1 CD44 CAR 7642 SEQ ID NO. 22 in WO2016042461A1 CD44 CAR 7643 SEQ ID NO. 23 in WO2016042461A1 CD44 CAR 7644 SEQ ID NO. 24 in WO2016042461A1 CD44 CAR 7645 SEQ ID NO. 25 in WO2016042461A1 CD44 CAR 7646 SEQ ID NO. 26 in WO2016042461A1 CD44 CAR 7647 SEQ ID NO. 27 in WO2016042461A1 CD44 CAR 7648 SEQ ID NO. 28 in WO2016042461A1 CD44 CAR 7649 SEQ ID NO. 31 in WO2016042461A1 CD44 CAR 7650 SEQ ID NO. 32 in WO2016042461A1 CD44 CAR 7651 SEQ ID NO. 33 in WO2016042461A1 CD44 CAR 7652 SEQ ID NO. 34 in WO2016042461A1 CD44 CAR 7653 SEQ ID NO. 35 in WO2016042461A1 CD4-DDY3 CAR 7654 SEQ ID NO: 9 in EP3074419A2 CD5 CAR 7655 SEQ ID NO. 15 in WO2016138491 CD5 CAR 7656 SEQ ID NO: 13 in WO2016172606A1 CD52 CAR 7657 SEQ ID NO. 18 in WO2016138491 CD7 CAR 7658 SEQ ID NO. 17 in WO2016138491 CD70 CAR 7659 SEQ ID NO. 100 in WO2015121454 CD70 CAR 7660 SEQ ID NO. 93 in WO2015121454 CD70 CAR 7661 SEQ ID NO. 94 in WO2015121454 CD70 CAR 7662 SEQ ID NO. 96 in WO2015121454 CD70 CAR 7663 SEQ ID NO. 101 in WO2015121454 CD70 CAR 7664 SEQ ID NO. 95 in WO2015121454 CD70 CAR 7665 SEQ ID NO. 97 in WO2015121454 CD70 CAR 7666 SEQ ID NO. 98 in WO2015121454 CD8 stalk APRIL CAR 7667 SEQ ID NO: 51 in US20160296562A1 CEA CAR 7668 SEQ ID NO. 4 in WO2016008973A1 CEA CAR 7669 SEQ ID NO. 29 in US20140242701A CEA CAR 7670 SEQ ID NO. 30 in US20140242701A Chimeric VNARCAR 1 7671 SEQ ID NO: 105 in US20160333094A1 Chimeric VNARCAR 2 7672 SEQ ID NO: 106 in US20160333094A1 Chimeric VNARCAR 3 7673 SEQ ID NO: 107 in US20160333094A1 Chimeric VNARCAR 4 7674 SEQ ID NO: 108 in US20160333094A1 Chimeric VNARCAR 5 7675 SEQ ID NO: 109 in US20160333094A1 Chimeric VNARCAR 6 7676 SEQ ID NO: 110 in US20160333094A1 CLDN6 CAR 7677 SEQ ID NO. 22 in WO2016150400 CLDN6 CAR 7678 SEQ ID NO. 23 in WO2016150400 CLDN6 CAR 7679 SEQ ID NO. 24 in WO2016150400 CLL1 CAR 7680 SEQ ID NO: 148 in WO2016179319A1 CLL1 CAR 7681 SEQ ID NO. 35 in WO2016120218 CLL1 CAR 7682 SEQ ID NO. 36 in WO2016120218 CLL1 CAR 7683 SEQ ID NO. 37 in WO2016120218 CLL1 CAR 7684 SEQ ID NO. 38 in WO2016120218 CLL1 CAR 7685 SEQ ID NO. 39 in WO2016120218 CLL1 CAR 7686 SEQ ID NO. 40 in WO2016120218 CLL1 CAR 7687 SEQ ID NO. 41 in WO2016120218 CLL1 CAR 7688 SEQ ID NO. 42 in WO2016120218 CLL1 CAR 7689 SEQ ID NO. 43 in WO2016120218 CLL1 CAR 7690 SEQ ID NO. 44 in WO2016120218 CLL1 CAR 7691 SEQ ID NO. 45 in WO2016120218 CLL1 CAR 7692 SEQ ID NO. 46 in WO2016120218 CLL1 CAR 7693 SEQ ID NO. 47 in WO2016120218 CLL1 CAR 7694 SEQ ID NO. 48 in WO2016120218 CLL1 CAR 7695 SEQ ID NO. 49 in WO2016120218 CLL1 CAR 7696 SEQ ID NO. 50 in WO2016120218 CLL1 CAR 7697 SEQ ID NO. 51 in WO2016120218 CLL1 CAR 7698 SEQ ID NO. 52 in WO2016120218 CLL1 CAR 7699 SEQ ID NO. 53 in WO2016120218 CLL1 CAR 7700 SEQ ID NO. 54 in WO2016120218 CLL1 CAR 7701 SEQ ID NO. 55 in WO2016120218 CLL1 CAR 7702 SEQ ID NO. 56 in WO2016120218 CLL1 CAR 7703 SEQ ID NO. 57 in WO2016120218 CLL1 CAR 7704 SEQ ID NO. 58 in WO2016120218 CLL1 CAR 7705 SEQ ID NO. 59 in WO2016120218 CLL1 CAR 7706 SEQ ID NO. 60 in WO2016120218 CLL1 CAR 7707 SEQ ID NO. 61 in WO2016120218 CLL1 CAR 7708 SEQ ID NO. 62 in WO2016120218 CLL1 CAR 7709 SEQ ID NO. 63 in WO2016120218 CLL1 CAR 7710 SEQ ID NO. 64 in WO2016120218 CLL1 CAR 7711 SEQ ID NO. 65 in WO2016120218 CLL1 CAR 7712 SEQ ID NO. 66 in WO2016120218 CLL1 CAR 7713 SEQ ID NO. 67 in WO2016120218 CLL1 CAR 7714 SEQ ID NO. 68 in WO2016120218 CLL1 CAR 7715 SEQ ID NO. 69 in WO2016120218 CLL1 CAR 7716 SEQ ID NO. 70 in WO2016120218 CLL1 CAR 7717 SEQ ID NO. 71 in WO2016120218 CLL1 CAR 7718 SEQ ID NO. 72 in WO2016120218 CLL1 CAR 7719 SEQ ID NO. 73 in WO2016120218 CLL1 CAR 7720 SEQ ID NO. 74 in WO2016120218 CLL1 CAR 7721 SEQ ID NO. 75 in WO2016120218 CLL1 CAR 7722 SEQ ID NO. 76 in WO2016120218 CLL1 CAR 7723 SEQ ID NO. 77 in WO2016120218 CLL1 CAR 7724 SEQ ID NO. 78 in WO2016120218 CLL1 CAR 7725 SEQ ID NO. 79 in WO2016120218 CLL1 CAR 7726 SEQ ID NO. 80 in WO2016120218 CLL1 CAR 7727 SEQ ID NO. 81 in WO2016120218 CLL1 CAR 7728 SEQ ID NO. 82 in WO2016120218 CLL1 CAR 7729 SEQ ID NO. 83 in WO2016120218 CLL1 CAR 7730 SEQ ID NO. 84 in WO2016120218 CLL1 CAR 7731 SEQ ID NO. 85 in WO2016120218 CLL1 CAR 7732 SEQ ID NO. 86 in WO2016120218 CLL1 CAR 7733 SEQ ID NO. 87 in WO2016120218 CLL1 CAR 7734 SEQ ID NO. 88 in WO2016120218 CLL1 CAR 7735 SEQ ID NO. 89 in WO2016120218 CLL1 CAR 7736 SEQ ID NO. 90 in WO2016120218 CLL1 CAR 7737 SEQ ID NO. 91 in WO2016120218 CLL1 CAR 7738 SEQ ID NO. 92 in WO2016120218 CLL1 CAR 7739 SEQ ID NO. 93 in WO2016120218 CLL1 CAR 7740 SEQ ID NO. 94 in WO2016120218 CLL1 CAR 7741 SEQ ID NO. 95 in WO2016120218 CLL1 CAR 7742 SEQ ID NO. 96 in WO2016120218 CLL1 CAR 7743 SEQ ID NO. 97 in WO2016120218 CLL1 CAR 7744 SEQ ID NO. 98 in WO2016120218 CLL1 CAR 7745 SEQ ID NO. 99 in WO2016120218 CLL1 CAR 7746 SEQ ID NO. 100 in WO2016120218 CLL1 CAR 7747 SEQ ID NO. 101 in WO2016120218 CLL1 CAR 7748 SEQ ID NO. 102 in WO2016120218 CLL1 CAR 7749 SEQ ID NO. 103 in WO2016120218 CLL1 CAR 7750 SEQ ID NO. 104 in WO2016120218 CLL1 CAR 7751 SEQ ID NO. 105 in WO2016120218 CLL1 CAR 7752 SEQ ID NO. 106 in WO2016120218 CLL1 CAR 7753 SEQ ID NO. 107 in WO2016120218 CLL1 CAR 7754 SEQ ID NO. 108 in WO2016120218 CLL1 CAR 7755 SEQ ID NO. 109 in WO2016120218 CLL1 CAR 7756 SEQ ID NO. 110 in WO2016120218 CLL1 CAR 7757 SEQ ID NO. 111 in WO2016120218 CLL1 CAR 7758 SEQ ID NO. 112 in WO2016120218 CLL1 CAR 7759 SEQ ID NO. 91 in WO2016014535 CLL1 CAR 7760 SEQ ID NO. 92 in WO2016014535 CLL1 CAR 7761 SEQ ID NO. 93 in WO2016014535 CLL1 CAR 7762 SEQ ID NO. 94 in WO2016014535 CLL1 CAR 7763 SEQ ID NO. 95 in WO2016014535 CLL1 CAR 7764 SEQ ID NO. 96 in WO2016014535 CLL1 CAR 7765 SEQ ID NO. 97 in WO2016014535 CLL1 CAR 7766 SEQ ID NO. 98 in WO2016014535 CLL1 CAR 7767 SEQ ID NO. 99 in WO2016014535 CLL1 CAR 7768 SEQ ID NO. 100 in WO2016014535 CLL1 CAR 7769 SEQ ID NO. 101 in WO2016014535 CLL1 CAR 7770 SEQ ID NO. 102 in WO2016014535 CLL1 CAR 7771 SEQ ID NO. 103 in WO2016014535 CLL1 CAR 7772 SEQ ID NO. 197 in WO2016014535 COM22 CAR 7773 SEQ ID NO: 358 in US20160297884A1 COM22 CAR 7774 SEQ ID NO: 359 in US20160297884A1 COM22 CAR 7775 SEQ ID NO: 360 in US20160297884A1 CS1 CAR 7776 SEQ ID No. 55 in WO2015121454 CS1 CAR 7777 SEQ ID No. 57 in WO2015121454 CS1 CAR 7778 SEQ ID No. 60 in WO2015121454 CS1 CAR 7779 SEQ ID No. 54 in WO2015121454 CS1 CAR 7780 SEQ ID No. 56 in WO2015121454 CS1 CAR 7781 SEQ ID No. 48 in WO2015121454 CS1 CAR 7782 SEQ ID No. 49 in WO2015121454 CS1 CAR 7783 SEQ ID No. 50 in WO2015121454 CS1 CAR 7784 SEQ ID No. 51 in WO2015121454 CS1 CAR 7785 SEQ ID No. 52 in WO2015121454 CS1 CAR 7786 SEQ ID No. 53 in WO2015121454 CS1 CAR 7787 SEQ ID No. 58 in WO2015121454 CS1 CAR 7788 SEQ ID No. 59 in WO2015121454 CS1 CAR 7789 SEQ ID No. 61 in WO2015121454 CS1 CAR 7790 SEQ ID No. 62 in WO2015121454 CS1 CAR 7791 SEQ ID NO. 28 in WO2014179759A1 DDD1/AD1 based zip CAR 7792 SEQ ID NO. 36 in WO2016124930 DDD1/AD1 Zip CAR 7793 SEQ ID NO. 37 in WO2016124930 EGFR CAR 7794 SEQ ID NO. 3 in WO2014130657 EGFR CAR 7795 SEQ ID NO. 2 in WO2014130657 EGFR CAR 7796 SEQ ID NO. 36 in US20140242701A EGFR CAR 7797 SEQ ID NO. 37 in US20140242701A EGFR CAR 7798 SEQ ID NO. 38 in US20140242701A EGFR CAR 7799 SEQ ID NO. 39 in US20140242701A EGFR CAR 7800 SEQ ID NO. 35 in US20140242701A EGFR CAR 7801 SEQ ID NO. 43 in WO2014130657 EGFR CAR 7802 SEQ ID NO. 96 in WO2014130657 EGFR CAR 7803 SEQ ID NO. 49 in WO2014130657 EGFR CAR 7804 SEQ ID NO. 55 in WO2014130657 EGFR CAR 7805 SEQ ID NO. 61 in WO2014130657 EGFR CAR 7806 SEQ ID NO. 67 in WO2014130657 EGFR CAR 7807 SEQ ID NO. 73 in WO2014130657 EGFR CAR 7808 SEQ ID NO. 79 in WO2014130657 EGFR CAR 7809 SEQ ID NO. 85 in WO2014130657 EGFR CAR 7810 SEQ ID NO. 90 in WO2014130657 EGFR CAR 7811 SEQ ID NO. 1 in WO2014130657 EGFR vIII CAR 7812 SEQ ID NO. 15 in WO2016016341 EGFR vIII CAR 7813 SEQ ID NO. 16 in WO2016016341 EGFR vIII CAR 7814 SEQ ID NO. 17 in WO2016016341 EGFR vIII CAR 7815 SEQ ID NO. 18 in WO2016016341 EGFR vIII CAR 7816 SEQ ID NO. 24 in WO2016016341 EGFR vIII CAR 7817 SEQ ID NO. 25 in WO2016016341 EGFR vIII CAR 7818 SEQ ID NO. 26 in WO2016016341 EGFR vIII CAR 7819 SEQ ID NO. 27 in WO2016016341 EGFR vIII CAR 7820 SEQ ID NO: 5 in US20160311907A1 EGFR vIII CAR 7821 SEQ ID NO: 10 in US20160311907A1 EGFR vIII CAR 7822 SEQ ID NO: 12 in US20160311907A1 EGFR vIII CAR 7823 SEQ ID NO: 8 in US20160311907A1 EGFR vIII CAR 7824 SEQ ID NO: 31 in US20160311907A1 EGFR vIII CAR 7825 SEQ ID NO: 30 in US20160311907A1 EGFR vIII CAR 7826 SEQ ID NO: 3 in US20160311907A1 EGFR vIII CAR 7827 SEQ ID NO: 10 in US20160200819A1 EGFRvIII CAR 7828 SEQ ID NO: 43 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7829 SEQ ID NO: 49 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7830 SEQ ID NO: 55 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7831 SEQ ID NO: 61 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7832 SEQ ID NO: 67 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7833 SEQ ID NO: 73 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7834 SEQ ID NO: 79 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7835 SEQ ID NO: 85 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7836 SEQ ID NO: 90 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7837 SEQ ID NO: 96 in U.S. Pat. No. 9,394,368B2 EGFRvIII CAR 7838 SEQ ID NO. 49 in US20170008963A1 EGFRvIII CAR 7839 SEQ ID NO. 55 in US20170008963A1 EGFRvIII CAR 7840 SEQ ID NO. 61 in US20170008963A1 EGFRvIII CAR 7841 SEQ ID NO. 67 in US20170008963A1 EGFRvIII CAR 7842 SEQ ID NO. 73 in US20170008963A1 EGFRvIII CAR 7843 SEQ ID NO. 79 in US20170008963A1 EGFRvIII CAR 7844 SEQ ID NO. 85 in US20170008963A1 EGFRvIII CAR 7845 SEQ ID NO. 90 in US20170008963A1 EGFRvIII CAR 7846 SEQ ID NO. 10 in US20140037628 EGFRvIII CAR 7847 SEQ ID NO. 11 in US20140037628 EGFRvIII CAR 7848 SEQ ID NO. 2 in US20170008963A1 EGFRvIII CAR 7849 ″EGFRvIII scFv 7850 SEQ ID NO. 1 in US20170008963A1 EGFRvIII scFv 7851 FcRL5 CAR 7852 SEQ ID NO. 11 in US20170008963A1 Folate Receptor CAR 7853 Folate Receptor CAR 7854 SEQ ID NO. 12 in US20170008963A1 Fra CAR 7855 SEQ ID NO: 959 (WO2016090337) Fra CAR 7856 SEQ ID NO. 13 in US20170002072A1 FRβ CAR 7857 SEQ ID NO. 22 in US20170002072A1 FRβ CAR 7858 SEQ ID NO: 13 in U.S. Pat. No. 9,402,865B2 FRβ CAR 7859 SEQ ID NO: 22 in U.S. Pat. No. 9,402,865B2 FRβ CAR 7860 SEQ ID NO: 2 in U.S. Pat. No. 9,446,105B2 FRβ CAR 7861 SEQ ID NO: 4 in U.S. Pat. No. 9,446,105B2 FRβ CAR 7862 SEQ ID NO: 6 in U.S. Pat. No. 9,446,105B2 GCN4 CAR 7863 SEQ ID NO: 8 in U.S. Pat. No. 9,446,105B2 GCN4 CAR 7864 SEQ ID NO: 10 in U.S. Pat. No. 9,446,105B2 GD2 CAR 7865 SEQ ID NO: 12 in U.S. Pat. No. 9,446,105B2 GD2 CAR 7866 SEQ ID NO: 273 in WO2016168773A3 GD2 CAR 7867 SEQ ID NO: 274 in WO2016168773A3 GD2 CAR 7868 SEQ ID No. 26 in WO2015132604 GD2 CAR 7869 SEQ ID No. 27 in WO2015132604 GD2 CAR 7870 SEQ ID No. 28 in WO2015132604 GD2 CAR 7871 SEQ ID No. 29 in WO2015132604 GD2 CAR 7872 SEQ ID No. 30 in WO2015132604 GD2 CAR 7873 SEQ ID No. 31 in WO2015132604 GD2 CAR 7874 SEQ ID No. 32 in WO2015132604 GD2 CAR 7875 SEQ ID No. 33 in WO2015132604 GD2 CAR 7876 SEQ ID No. 34 in WO2015132604 GD2 CAR 7877 SEQ ID No. 35 in WO2015132604 GD2 CAR 7878 SEQ ID No. 36 in WO2015132604 GD2 CAR 7879 SEQ ID No. 37 in WO2015132604 GD2 CAR 7880 in WO2016134284 (no SEQ ID NO) GD3 CAR 7881 in WO2016134284 (no SEQ ID NO) GD3 CAR 7882 in WO2016134284 (no SEQ ID NO) GD3 CAR 7883 in WO2016134284 (no SEQ ID NO) GD3 CAR 7884 SEQ ID NO: 19 in WO2016185035A1 GD3 CAR 7885 SEQ ID NO: 20 in WO2016185035A1 GD3 CAR 7886 SEQ ID NO: 21 in WO2016185035A1 GD3 CAR 7887 SEQ ID NO: 22 in WO2016185035A1 GD3 CAR 7888 SEQ ID NO: 23 in WO2016185035A1 GD3 CAR 7889 SEQ ID NO: 24 in WO2016185035A1 GD3 CAR 7890 SEQ ID NO: 25 in WO2016185035A1 GD3 CAR 7891 SEQ ID NO: 26 in WO2016185035A1 GFRalpha CAR 7892 SEQ ID NO: 27 in WO2016185035A1 GPC3 CAR 7893 SEQ ID NO: 28 in WO2016185035A1 GPC3 CAR 7894 SEQ ID NO: 29 in WO2016185035A1 GPC3 CAR 7895 GPC3 CAR 7896 SEQ ID NO. 3 in WO2016049459 GPC3 CAR 7897 SEQ ID NO. 27 in WO2016049459 GPC3 CAR 7898 SEQ ID NO. 10 in WO2016049459 GPC3 CAR 7899 SEQ ID NO. 29 in WO2016049459 GPC3 CAR 7900 SEQ ID NO. 14 in WO2016049459 GPC3 CAR 7901 SEQ ID NO. 30 in WO2016049459 GPC3CAR 7902 SEQ ID NO. 31 in WO2016049459 GPC3CAR 7903 SEQ ID NO. 18 in WO2016049459 GPC3CAR 7904 SEQ ID NO. 33 in WO2016049459 GPC3CAR 7905 SEQ ID NO: 22 in US20160215261A1 Her1/Her3 CAR Bispecific 7906 SEQ ID NO: 23 in US20160215261A1 Her1/Her3 CAR Bispecific 7907 SEQ ID NO: 24 in US20160215261A1 HER2 CAR 7908 SEQ ID NO: 25 in US20160215261A1 HER2 CAR 7909 SEQ ID NO: 9 of WO2016073629 HER2 CAR 7910 SEQ ID NO: 10 of WO2016073629 HER2 CAR 7911 SEQ ID NO: 17 in US20160333114A1 HER2 CAR 7912 SEQ ID NO: 28 in US20160333114A1 HER2 CAR 7913 SEQ ID NO: 98 in US20160333114A1 HER2 CAR 7914 SEQ ID NO: 110 in US20160333114A1 HER2 CAR 7915 SEQ ID NO: 271 in WO2016168773A3 HER2 CAR 7916 SEQ ID NO: 272 in WO2016168773A3 HER2 CAR 7917 SEQ ID NO: 5 in WO2016168769A1 HERVK CAR 7918 SEQ ID NO: 6 in WO2016168769A1 HIV Env CAR 7919 SEQ ID NO: 48 in WO2016168766A1 HIV Env CAR 7920 SEQ ID NO: 49 in WO2016168766A1 HIV Env CAR 7921 SEQ ID NO: 4 in EP2997134A4 HIV Env CAR 7922 SEQ ID NO. 7 in WO2015077789 HIV Env CAR 7923 SEQ ID NO. 9 in WO2015077789 HIV Env CAR 7924 SEQ ID NO. 47 in WO2015077789 HIV Env CAR 7925 SEQ ID NO. 49 in WO2015077789 HSP70 CAR 7926 SEQ ID NO. 51 in WO2015077789 HSP70 CAR 7927 SEQ ID NO. 53 in WO2015077789 HSP70 CAR 7928 SEQ ID NO. 5 in WO2015077789 HSP70 CAR 7929 SEQ ID NO. 21 in WO2016120217 HSP70 CAR 7930 SEQ ID NO. 22 in WO2016120217 HSP70 CAR 7931 SEQ ID NO. 23 in WO2016120217 HSP70 CAR 7932 SEQ ID NO. 24 in WO2016120217 HSP70 CAR 7933 SEQ ID NO. 25 in WO2016120217 HSP70 CAR 7934 SEQ ID NO. 26 in WO2016120217 HSP70 CAR 7935 SEQ ID NO. 27 in WO2016120217 HSP70 CAR 7936 SEQ ID NO. 28 in WO2016120217 HSP70 CAR 7937 SEQ ID NO. 29 in WO2016120217 IL 13 CAR 7938 SEQ ID NO. 30 in WO2016120217 IL 13 CAR 7939 SEQ ID NO. 31 in WO2016120217 IL 13 CAR 7940 SEQ ID NO. 32 in WO2016120217 IL13Ra2specific CAR 7941 SEQ ID NO. 4 in WO2016089916A1 IL13Ra2specific CAR 7942 SEQ ID NO. 5 in WO2016089916A1 IL13Ra2specific CAR 7943 SEQ ID NO. 6 in WO2016089916A1 IL13Ra2specific CAR 7944 SEQ ID NO. 47 in WO2016123143 IL13Ra2specific CAR 7945 SEQ ID NO. 49 in WO2016123143 IL13Rα2 CAR 7946 SEQ ID NO. 51 in WO2016123143 IL13Rα2 CAR 7947 SEQ ID NO. 53 in WO2016123143 IL13Rα2 CAR 7948 SEQ ID NO. 55 in WO2016123143 IL13Rα2 CAR 7949 SEQ ID NO: 1 in US20160340649A1 IL13Rα2 CAR 7950 SEQ ID NO: 31 in US20160340649A1 IL13Rα2 CAR 7951 SEQ ID NO: 32 in US20160340649A1 IL13Rα2 CAR 7952 SEQ ID NO: 33 in US20160340649A1 IL13Rα2 CAR 7953 SEQ ID NO: 34 in US20160340649A1 IL13Rα2 CAR 7954 SEQ ID NO: 35 in US20160340649A1 IL13Rα2 CAR 7955 SEQ ID NO: 36 in US20160340649A1 IL13Rα2 CAR 7956 SEQ ID NO: 37 in US20160340649A1 IL13Rα2 CAR 7957 SEQ ID NO: 38 in US20160340649A1 IL13Rα2 CAR 7958 SEQ ID NO: 39 in US20160340649A1 IL13Rα2 CAR 7959 SEQ ID NO: 40 in US20160340649A1 IL13Rα2 CAR 7960 SEQ ID NO: 41 in US20160340649A1 IL13Rα2 CAR 7961 SEQ ID NO: 42 in US20160340649A1 IL13Rα2 CAR 7962 SEQ ID NO: 43 in US20160340649A1 IL13Rα2 CAR 7963 SEQ ID NO: 44 in US20160340649A1 IL13Rα2 CAR 7964 SEQ ID NO: 45 in US20160340649A1 KMA CAR 7965 SEQ ID NO: 46 in US20160340649A1 MESOTHELIN CAR 7966 SEQ ID NO: 47 in US20160340649A1 MESOTHELIN CAR 7967 SEQ ID NO: 48 in US20160340649A1 MESOTHELIN CAR 7968 SEQ ID NO: 27 in WO2016172703A2 MESOTHELIN CAR 7969 SEQ ID NO: 18 in WO2013142034 MESOTHELIN CAR 7970 SEQ ID NO: 19 in WO2013142034 MESOTHELIN CAR 7971 SEQ ID NO: 20 in WO2013142034 MESOTHELIN CAR 7972 SEQ ID NO: 21 in WO2013142034 MESOTHELIN CAR 7973 SEQ ID NO: 22 in WO2013142034 MESOTHELIN CAR 7974 SEQ ID NO: 23 in WO2013142034 Mesothelin CAR 7975 SEQ ID NO. 3 in WO2013067492 MUC1 CAR 7976 SEQ ID NO. 5 in WO2013063419 MUC1 CAR 7977 SEQ ID NO. 7 in WO2013063419 MUC1 CAR 7978 SEQ ID NO. 51 in US20160340406A1 MUC1 CAR 7979 SEQ ID NO. 30 in US20160130357 MUC1 CAR 7980 SEQ ID NO. 32 in US20160130357 MUC1 CAR 7981 SEQ ID NO. 34 in US20160130357 MUC1 CAR 7982 SEQ ID NO. 295 in WO2016130726 MUC1 CAR 7983 SEQ ID NO. 298 in WO2016130726 MUC1 CAR 7984 SEQ ID NO. 301 in WO2016130726 MUC1 CAR 7985 SEQ ID NO. 304 in WO2016130726 MUC1 CAR 7986 SEQ ID NO. 307 in WO2016130726 MUC1 CAR 7987 SEQ ID NO. 607 in WO2016130726 MUC1 CAR 7988 SEQ ID NO. 609 in WO2016130726 MUC1 CAR 7989 SEQ ID NO. 611 in WO2016130726 MUC1 CAR 7990 SEQ ID NO. 613 in WO2016130726 NCAR with RQR82ACD19CAR 7991 SEQ ID NO. 615 in WO2016130726 NYBR1 CAR 7992 SEQ ID NO. 617 in WO2016130726 NYBR1 CAR 7993 SEQ ID NO. 619 in WO2016130726 NYBR1 CAR 7994 SEQ ID NO: 218 in WO2016097231 NYBR1 CAR 7995 SEQ ID NO. 26 in WO2015112830 NYBR1 CAR 7996 SEQ ID NO. 29 in WO2015112830 NYBR1 CAR 7997 SEQ ID NO. 60 in WO2015112830 NYBR1 CAR 7998 SEQ ID NO: 1 in US20160333422A1 P5A CAR 7999 SEQ ID NO: 26 in US20160333422A1 P5A CAR 8000 SEQ ID NO: 29 in US20160333422A1 P5A CAR 8001 SEQ ID NO: 60 in US20160333422A1 P5AC1 CAR 8002 SEQ ID NO: 343 in US20160297884A1 P5AC1 CAR 8003 SEQ ID NO: 344 in US20160297884A1 P5AC1 CAR 8004 SEQ ID NO: 345 in US20160297884A1 P5AC1 CAR 8005 SEQ ID NO: 346 in US20160297884A1 P5AC16 CAR 8006 SEQ ID NO: 347 in US20160297884A1 P5AC16 CAR 8007 SEQ ID NO: 396 in US20160297884A1 P5AC16 CAR 8008 SEQ ID NO: 348 in US20160297884A1 P6AP CAR 8009 SEQ ID NO: 349 in US20160297884A1 P6AP CAR 8010 SEQ ID NO: 350 in US20160297884A1 P6AP CAR 8011 SEQ ID NO: 351 in US20160297884A1 P6DY CAR 8012 SEQ ID NO: 364 in US20160297884A1 P6DY CAR 8013 SEQ ID NO: 365 in US20160297884A1 P6DY CAR 8014 SEQ ID NO: 366 in US20160297884A1 PC1 CAR 8015 SEQ ID NO: 361 in US20160297884A1 PC1 CAR 8016 SEQ ID NO: 362 in US20160297884A1 PC1 CAR 8017 SEQ ID NO: 363 in US20160297884A1 PC1C12 CAR 8018 SEQ ID NO: 352 in US20160297884A1 PC1C12 CAR 8019 SEQ ID NO: 353 in US20160297884A1 PC1C12 CAR 8020 SEQ ID NO: 354 in US20160297884A1 PD1 CAR 8021 SEQ ID NO: 355 in US20160297884A1 PD1 CAR 8022 SEQ ID NO: 356 in US20160297884A1 PD1 CAR 8023 SEQ ID NO: 357 in US20160297884A1 PD1 CAR 8024 SEQ ID NO. 119 in WO2014153270 PD1 CAR 8025 SEQ ID NO. 121 in WO2014153270 PD1 CAR 8026 SEQ ID NO: 22 in US20160311917A1 PD1 CAR 8027 SEQ ID NO: 24 in US20160311917A1 PD1 CAR 8028 SEQ ID NO: 63 in US20160311917A1 PD1 CAR 8029 SEQ ID NO: 64 in US20160311917A1 PD1 CAR 8030 SEQ ID NO: 65 in US20160311917A1 PD1 CAR 8031 SEQ ID NO: 66 in US20160311917A1 PD1 CAR 8032 SEQ ID NO: 67 in US20160311917A1 PD1 CAR 8033 SEQ ID NO: 68 in US20160311917A1 PD1 CAR 8034 SEQ ID NO: 69 in US20160311917A1 PD1 CAR 8035 SEQ ID NO: 70 in US20160311917A1 PD1 CAR 8036 SEQ ID NO: 71 in US20160311917A1 PD1 CAR 8037 SEQ ID NO: 72 in US20160311917A1 PD1 CAR 8038 SEQ ID NO: 73 in US20160311917A1 PD1 CAR 8039 SEQ ID NO: 74 in US20160311917A1 PD1 CAR 8040 SEQ ID NO: 75 in US20160311917A1 PD1 CAR 8041 SEQ ID NO: 76 in US20160311917A1 PD1 CAR 8042 SEQ ID NO: 77 in US20160311917A1 PD1 CAR 8043 SEQ ID NO: 78 in US20160311917A1 PD1 CAR 8044 SEQ ID NO: 79 in US20160311917A1 PD1 CAR 8045 SEQ ID NO: 80 in US20160311917A1 PD1 CAR 8046 SEQ ID NO: 81 in US20160311917A1 PD1 CAR 8047 SEQ ID NO: 82 in US20160311917A1 PD1 CAR 8048 SEQ ID NO: 83 in US20160311917A1 PD1 CAR 8049 SEQ ID NO: 84 in US20160311917A1 PD1 CAR 8050 SEQ ID NO: 85 in US20160311917A1 PD1 CAR 8051 SEQ ID NO: 86 in US20160311917A1 PD1 CAR 8052 SEQ ID NO: 26 in WO2016172537A1 PD1 CAR 8053 SEQ ID NO: 39 in WO2016172537A1 PD1 CAR 8054 SEQ ID NO: 40 in US20160311907A1 PD1 CAR 8055 SEQ ID. NO. 121 in WO2015157252 PD1 CAR 8056 SEQ ID. NO. 119 in WO2015157252 PD1 CAR 8057 SEQ ID NO. 24 (WO2016014565) PD1 CAR 8058 SEQ ID NO. 22 (WO2016014565) PD1 FKBP RCAR 8059 SEQ ID NO. 23 (WO2016014565) PD1 FKBP RCAR 8060 SEQ ID NO. 26 in WO2015142675 PSMA NCAR 8061 SEQ ID NO. 39 in WO2015142675 PSMA NCAR 8062 SEQ ID NO: 28 in US20160311907A1 PSMA NCAR 8063 SEQ ID NO: 29 in US20160311907A1 PSMA NCAR 8064 SEQ ID NO: 140 in WO2016097231 PSMA NCAR 8065 SEQ ID NO: 144 in WO2016097231 PSMA NCAR 8066 SEQ ID NO: 145 in WO2016097231 PSMA NCAR 8067 SEQ ID NO: 146 in WO2016097231 PSMA NCAR 8068 SEQ ID NO: 147 in WO2016097231 PSMA NCAR 8069 SEQ ID NO: 148 in WO2016097231 PSMA NCAR 8070 SEQ ID NO: 149 in WO2016097231 PSMA NCAR 8071 SEQ ID NO: 150 in WO2016097231 PSMA NCAR 8072 SEQ ID NO: 167 in WO2016097231 PSMA NCAR 8073 SEQ ID NO: 168 in WO2016097231 PSMA NCAR 8074 SEQ ID NO. 169 in WO2016097231 PSMA NCAR 8075 SEQ ID NO: 170 in WO2016097231 PSMA NCAR 8076 SEQ ID NO: 171 in WO2016097231 PSMA NCAR 8077 SEQ ID NO: 172 in WO2016097231 PSMA NCAR 8078 SEQ ID NO: 173 in WO2016097231 PSMA NCAR 8079 SEQ ID NO: 174 in WO2016097231 PSMA NCAR 8080 SEQ ID NO: 151 in WO201609723I PSMA NCAR 8081 SEQ ID NO: 152 in WO2016097231 PSMA NCAR 8082 SEQ ID NO: 153 in WO2016097231 PSMA NCAR 8083 SEQ ID NO: 154 in WO2016097231 PSMA NCAR 8084 SEQ ID NO: 155 in WO2016097231 PSMA NCAR 8085 SEQ ID NO: 156 in WO2016097231 PSMA NCAR 8086 SEQ ID NO: 139 in WO2016097231 PSMA NCAR 8087 SEQ ID NO: 138 in WO2016097231 PSMA NCAR 8088 SEQ ID NO: 175 in WO2016097231 PSMA NCAR 8089 SEQ ID NO: 157 in WO2016097231 PSMA NCAR 8090 SEQ ID NO: 158 in WO2016097231 PSMA NCAR 8091 SEQ ID NO: 159 in WO2016097231 PSMA NCAR 8092 SEQ ID NO: 160 in WO2016097231 PSMA NCAR 8093 SEQ ID NO: 161 in WO2016097231 PSMA NCAR 8094 SEQ ID NO: 162 in WO2016097231 PSMA NCAR 8095 SEQ ID NO: 163 in WO2016097231 PSMA NCAR 8096 SEQ ID NO: 164 in WO2016097231 PSMA NCAR 8097 SEQ ID NO: 165 in WO2016097231 PSMA NCAR 8098 SEQ ID NO: 166 in WO2016097231 PSMA NCAR 8099 SEQ ID NO: 141 in WO2016097231 PSMA NCAR 8100 SEQ ID NO: 142 in WO2016097231 PSMA NCAR 8101 SEQ ID NO: 143 in WO2016097231 PSMA NCAR 8102 SEQ ID NO: 214 in WO2016097231 ROR1 CAR 8103 SEQ ID NO: 216 in WO2016097231 ROR1 CAR 8104 SEQ ID NO: 217 in WO2016097231 ROR1 CAR 8105 SEQ ID NO: 215 in WO2016097231 ROR1 CAR 8106 SEQ ID N0. 79 in WO2016016344A1 ROR1 CAR 8107 SEQ ID N0. 80 in WO2016016344A1 ROR1 CAR 8108 SEQ ID N0. 81 in WO2016016344A1 ROR1 CAR 8109 SEQ ID N0. 82 in WO2016016344A1 ROR1 CAR 8110 SEQ ID N0. 83 in WO2016016344A1 ROR1 CAR 8111 SEQ ID N0. 84 in WO2016016344A1 ROR1 CAR 8112 SEQ ID N0. 85 in WO2016016344A1 ROR1 CAR 8113 SEQ ID N0. 86 in WO2016016344A1 ROR1 CAR 8114 SEQ ID N0. 87 in WO2016016344A1 ROR1 CAR 8115 SEQ ID N0. 88 in WO2016016344A1 ROR1 CAR 8116 SEQ ID N0. 89 in WO2016016344A1 ROR1 CAR 8117 SEQ ID N0. 90 in WO2016016344A1 ROR1 CAR 8118 SEQ ID N0. 91 in WO2016016344A1 ROR1 CAR 8119 SEQ ID N0. 92 in WO2016016344A1 ROR1 CAR 8120 SEQ ID N0. 93 in WO2016016344A1 ROR1 CAR 8121 SEQ ID N0. 94 in WO2016016344A1 ROR1 CAR 8122 SEQ ID N0. 95 in WO2016016344A1 ROR1 CAR 8123 SEQ ID N0. 96 in WO2016016344A1 ROR1 CAR 8124 SEQ ID N0. 103 in WO2016016344A1 ROR1 CAR 8125 SEQ ID N0. 104 in WO2016016344A1 ROR1 CAR 8126 SEQ ID N0. 105 in WO2016016344A1 ROR1 CAR 8127 SEQ ID N0. 106 in WO2016016344A1 ROR1 CAR 8128 SEQ ID N0. 107 in WO2016016344A1 ROR1 CAR 8129 SEQ ID N0. 108 in WO2016016344A1 ROR1 CAR 8130 SEQ ID N0. 109 in WO2016016344A1 ROR1 CAR 8131 SEQ ID N0. 110 in WO2016016344A1 ROR1 CAR 8132 SEQ ID N0. 111 in WO2016016344A1 ROR1 CAR 8133 SEQ ID N0. 112 in WO2016016344A1 ROR1 CAR 8134 SEQ ID N0. 113 in WO2016016344A1 ROR1 CAR 8135 SEQ ID N0. 114 in WO2016016344A1 ROR1 CAR 8136 SEQ ID N0. 115 in WO2016016344A1 ROR1 CAR 8137 SEQ ID N0. 116 in WO2016016344A1 ROR1 CAR 8138 SEQ ID N0. 117 in WO2016016344A1 ROR1 CAR 8139 SEQ ID N0. 118 in WO2016016344A1 ROR1 CAR 8140 SEQ ID N0. 119 in WO2016016344A1 ROR1 CAR 8141 SEQ ID N0. 120 in WO2016016344A1 ROR1 CAR 8142 SEQ ID N0. 127 in WO2016016344A1 ROR1 CAR 8143 SEQ ID N0. 128 in WO2016016344A1 ROR1 CAR 8144 SEQ ID N0. 129 in WO2016016344A1 ROR1 CAR 8145 SEQ ID N0. 130 in WO2016016344A1 ROR1 CAR 8146 SEQ ID N0. 131 in WO2016016344A1 ROR1 CAR 8147 SEQ ID N0. 132 in WO2016016344A1 ROR1 CAR 8148 SEQ ID N0. 133 in WO2016016344A1 ROR1 CAR 8149 SEQ ID N0. 134 in WO2016016344A1 ROR1 CAR 8150 SEQ ID N0. 135 in WO2016016344A1 ROR1 CAR 8151 SEQ ID N0. 136 in WO2016016344A1 ROR1 CAR 8152 SEQ ID N0. 137 in WO2016016344A1 ROR1 CAR 8153 SEQ ID N0. 138 in WO2016016344A1 ROR1 CAR 8154 SEQ ID N0. 97 in WO2016016344A1 ROR1 CAR 8155 SEQ ID N0. 98 in WO2016016344A1 ROR1 CAR 8156 SEQ ID N0. 99 in WO2016016344A1 ROR1 CAR 8157 SEQ ID N0. 100 in WO2016016344A1 ROR1 CAR 8158 SEQ ID N0. 101 in WO2016016344A1 ROR1 CAR 8159 SEQ ID N0. 102 in WO2016016344A1 ROR1 CAR 8160 SEQ ID N0. 121 in WO2016016344A1 ROR1 CAR 8161 SEQ ID N0. 122 in WO2016016344A1 ROR1 CAR 8162 SEQ ID N0. 123 in WO2016016344A1 ROR1 CAR 8163 SEQ ID N0. 124 in WO2016016344A1 ROR1 CAR 8164 SEQ ID N0. 125 in WO2016016344A1 ROR1 CAR 8165 SEQ ID N0. 126 in WO2016016344A1 ROR1 CAR 8166 SEQ ID NO: 386 in WO2016187216A1 ROR1 CAR 8167 SEQ ID NO: 387 in WO2016187216A1 ROR1 CAR 8168 SEQ ID NO: 388 in WO2016187216A1 ROR1 CAR 8169 SEQ ID NO: 389 in WO2016187216A1 ROR1 CAR 8170 SEQ ID NO: 390 in WO2016187216A1 ROR1 CAR 8171 SEQ ID NO: 391 in WO2016187216A1 ROR1 CAR 8172 SEQ ID NO: 392 in WO2016187216A1 ROR1 CAR 8173 SEQ ID NO: 393 in WO2016187216A1 ROR1 CAR 8174 SEQ ID NO: 394 in WO2016187216A1 SNAP CAR 8175 SEQ ID NO: 395 in WO2016187216A1 SSEA4CAR 8176 SEQ ID NO: 396 in WO2016187216A1 SSEA4CAR 8177 SEQ ID NO: 397 in WO2016187216A1 Tan CAR (a CAR AND GATE which 8178 SEQ ID NO: 19 in US20160311907A1 recognizes CD19 AND CD33 using a CD45 phosphatase) Tan CAR (a CAR AND gate which 8179 SEQ ID NO. 5 in WO2016026742A1 recognizes CD19 AND CD33 using a CD148 phosphatase) Tan CAR (a CAR AND NOT gate 8180 SEQ ID NO. 6 in WO2016026742A1 which recognizes CD19 AND NOT CD33 and is based on an ITIM containing endodomain from LAIR1) Tan CAR (a CAR AND NOT GATE 8181 SEQ ID NO. 3 in WO2015075468 which recognizes CD19 AND NOT CD33 based on PTPN6 phosphatase) Tan CAR (a CAR AND NOT gate 8182 SEQ ID NO. 2 in WO2015075468 which recognizes CD19 AND NOT CD33 and recruits a PTPN6CD148 fusion protein to an ITIM containing endodomain) TOSO CAR 8183 SEQ ID NO. 5 in WO2015075468 TOSO CAR 8184 SEQ ID NO. 4 in WO2015075468 Trophoblast Glycoprotein 5T4 CAR 8185 SEQ ID NO. 6 in WO2015075468 Trophoblast Glycoprotein 5T4 CAR 8186 SEQ ID No. 4 in US20160347854A1 Trophoblast Glycoprotein 5T4 CAR 8187 SEQ ID No. 4 in EP3098237A1 Trophoblast Glycoprotein 5T4 CAR 8188 SEQ ID N0. 19 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8189 SEQ ID N0. 20 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8190 SEQ ID N0. 21 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8191 SEQ ID N0. 22 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8192 SEQ ID N0. 23 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8193 SEQ ID N0. 24 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8194 SEQ ID N0. 25 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8195 SEQ ID N0. 26 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8196 SEQ ID N0. 27 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8197 SEQ ID N0. 28 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8198 SEQ ID N0. 29 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8199 SEQ ID N0. 30 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8200 SEQ ID N0. 31 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8201 SEQ ID N0. 32 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8202 SEQ ID N0. 33 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8203 SEQ ID N0. 34 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8204 SEQ ID N0. 35 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8205 SEQ ID N0. 36 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8206 SEQ ID N0. 37 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8207 SEQ ID N0. 38 in WO2016034666A1 Trophoblast Glycoprotein 5T4 CAR 8208 SEQ ID N0. 39 in WO2016034666A1 TSLPR CAR 8209 SEQ ID N0. 40 in WO2016034666A1 TSLPR CAR 8210 SEQ ID N0. 41 in WO2016034666A1 TSLPR CAR 8211 SEQ ID N0. 42 in WO2016034666A1 TSLPR CAR 8212 SEQ ID NO. 39 in WO2015084513 TSLPR CAR 8213 SEQ ID NO. 40 in WO2015084513 TSLPR CAR 8214 SEQ ID NO. 41 in WO2015084513 TSLPR CAR 8215 SEQ ID NO. 42 in WO2015084513 TSLPR CAR 8216 SEQ ID NO. 43 in WO2015084513 TSLPR CAR 8217 SEQ ID NO. 44 in WO2015084513 TSLPR CAR 8218 SEQ ID NO. 45 in WO2015084513 TSLPR CAR 8219 SEQ ID NO. 46 in WO2015084513 TSLPR CAR 8220 SEQ ID NO: 39 in US20160311910A1 TSLPR CAR 8221 SEQ ID NO: 40 in US20160311910A1 TSLPR CAR 8222 SEQ ID NO: 41 in US20160311910A1 TSLPR CAR 8223 SEQ ID NO: 42 in US20160311910A1 TSLPR CAR 8224 SEQ ID NO: 43 in US20160311910A1 VEGFR2 CAR 8225 SEQ ID NO: 44 in US20160311910A1 VEGFR2 CAR 8226 SEQ ID NO: 45 in US20160311910A1 VEGFR2 CAR 8227 SEQ ID NO: 46 in US20160311910A1 VEGFR2 CAR 8228 SEQ ID NO. 10 in US20120213783 VEGFR2 CAR 8229 SEQ ID NO. 11 in US20120213783 VEGFR2 CAR 8230 SEQ ID NO. 12 in US20120213783 αfolate receptor (FRα) CAR 8231 SEQ ID NO. 13 in US20120213783 αfolate receptor (FRα) CAR 8232 SEQ ID NO. 14 in US20120213783

In one embodiment of the present invention, the payload of the invention is a CD19 specific CAR targeting different B cell malignancies and HER2-specific CAR targeting sarcoma, glioblastoma, and advanced Her2-positive lung malignancy.

In some embodiments, the CAR is a CD19 CAR. The amino acid sequences of CD19 CAR components and CD19 CAR constructs are presented in Table 17A and Table 17B. Table 17B also provides alternate aliases for a given construct ID. These aliases are identified by the prefix OT.

TABLE 17A CD19 CAR construct components AA NA SEQ SEQ Description Amino Acid Sequence ID NO ID NO CD19 scFv DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ 8233 8241- KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN 8246 LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGG GGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSR LTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGG SYAMDYWGQGTSVTVSS CD8a hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR 8234 8247- GLDFACD 8251 CD8a hinge- TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR 8235 8252- TM (hinge and GLDFACDIYIWAPLAGTCGVLLLSLVITLYC 8254 transmembrane) CD3 zeta RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD 8236 8255- signaling KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY 8261 domain SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE 8237 8262- intracellular GGCEL 8267 signaling domain; CD28 co-stimulatory domain; CD8α leader MALPVTALLLPLALLLHAARP  278  279-  283 hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL    3  339 (Amino acid CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKN 535-860 of VAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILA WT) LLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEH HHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILA TDLALYIKRRGEFFELIRKNQFNLEDPHQKELFLAML MTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKEL NIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHV SEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL   12  359 (Amino acid CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKN 535-860 of VAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILA WT, R732L) LLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEH HHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILA TDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAML MTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKEL NIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHV SEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL  505  520 (Amino acid CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKN 535-860 of VAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILA WT, R732L, LLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEH D764N) HHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILA TDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAML MTACNLSAITKPWPIQQRIAELVATEFFDQGDRERKEL NIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHV SEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL  227  233 (Amino acid CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKN 535-860 of VAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILA WT, R732L, LLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEH F736A) HHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILA TDLALYIKRLGEFAELIRKNQFNLEDPHQKELFLAML MTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKEL NIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHV SEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL  348  361 (Amino acid CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKN 535-860 of VAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILA WT, H653 A) LLIAALSADLDHRGVNNSYIQRSEHPLAQLYCHSIMEH HHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILA TDLALYIKRRGEFFELIRKNQFNLEDPHQKELFLAML MTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKEL NIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHV SEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL  509  524 (Amino acid CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKN 535-860 of VAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILA WT, R732L, LLIAALSADLDHRGVNNSYIQRSEHPLAQLYCHSIMEH H653A) HHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILA TDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAML MTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKEL NIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHV SEDCFPLLDGCRKNRQKWQALAEQQ hPDE5 EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETAL  510  525 (Amino acid CTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKN 535-860 of VAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILA WT, R732L, LLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEH D764A) HHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILA TDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAML MTACALSAITKPWPIQQRIAELVATEFFDQGDRERKEL NIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHV SEDCFPLLDGCRKNRQKWQALAEQQ HA Tag YPYDVPDYA 8238 8268 P2A Cleavable GATNFSLLKQAGDVEENPGP 8239 8270 peptide mCherry LSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEG 8240 8269 (M1L) EGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK AYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVT VTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMG WEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVK TTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYE RAEGRHSTGGMDELYK SG LINKER SG — AGTG GT Linker (GSG) GSG — GGAT (BamH1-Gly) CCGG A Flexible G/S GS — GGAT rich linker; CC BamH1 Site Lys-Asp Acid LD — CTAG Linker AT

TABLE 17B CD19 CARs constructs AA NA SEQ SEQ ID ID Description Amino Acid Sequence NO NO OT-CD 19-063 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8271 8285 (OT-001407) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFv; CD8a NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ Hinge and ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR Transmembmne KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF Domain; 4-1BB LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG intracellular TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG domain; CD3 zeta GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC signaling domain; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE stop GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR* OT-CD 19-037 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8272 8286 (OT-001258) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG Linker (GSG), GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5 (Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT, R732L, EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD D764N), stop KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRGSGEETRELQSLAAAVVPSAQTLK ITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMF AALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNS YIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQI LSGLSIEEYKTTLKIIKQAILADLALYIKRLGEFFELIR KNQFNLEDPHQKELFLAMLMTACNLSAITKPWPIQQ RIAELVAIEFFDQGDRERKELNIEPTDLMNREKKNKI PSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQ* OT-CD 19-045 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8273 8287 (OT-001298) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG Linker (GSG); GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5 (Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT, R732L, EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD F736A); stop KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRGSGEETRELQSLAAAVVPSAQTLK ITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMF AALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNS YIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQI LSGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFAELI RKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQ QRIAELVAIEFFDQGDRERKELNIEPTDLMNREKKN KIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRK NRQKWQALAEQQ* OT-CD19-051 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8274 8288 (OT-001299) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG Linker (GSG); GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5(Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT); stop EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRGSGEETRELQSLAAAVVPSAQTLK ITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKH EVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMF AALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNS YIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQI LSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFFELI RKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQ QRIAELVAIEFFDQGDRERKELNIEPTDLMNREKKN KIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRK NRQKWQALAEQQ* OT-CD 19-052 MALPVTALLLPLALLLHAARPYPYDVPDYADIQMTQ 8275 8289 (OT-001300) TTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT CD8a leader; HA VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQE Tag; CD19 scFV; DIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS CD8a-Tm; (4- GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY 1BB intracellular GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI domain); CD3 zeta IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY signaling domain; AMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSL Linker (SG); RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV hPDE5 (Amino LLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED acid 535-860 of GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL WT); stop YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPRSGEETRELQSLAAA VVPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNL VQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHA FNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDL DHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLM ILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKR RGEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAI TKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLM NREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPL LDGCRKNRQKWQALAEQQ* OT-CD 19-053 MALPVTALLLPLALLLHAARPYPYDVPDYADIQMTQ 8276 8290 (OT-001301) TTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT CD8a leader; HA VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQE Tag; CD19 scFV; DIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS CD8a-Tm; (4- GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY 1BB intracellular GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI domain); CD3 zeta IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY signaling domain; AMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSL linker (SG); RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV hPDE5 (Amino LLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED acid 535-860 of GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL WT, R732L); stop YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPRSGEETRELQSLAAA VVPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNL VQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHA FNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDL DHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLM ILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKR LGEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAI TKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLM NREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPL LDGCRKNRQKWQALAEQQ* OT-CD 19-067 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8277 8291 (OT-001302) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG linker (GS); GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5 (Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT, R732L, EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD F736A); linker KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT (GS); mCherry YDALHMQALPPRGSEETRELQSLAAAVVPSAQTLKI (M1L); stop TDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHE VLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS GLSIEEYKTTLKIIKQAILATDLALYIKRLGEFAELIRK NQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRI AELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ KWQALAEQQGSLSKGEEDNMAIIKEFMRFKVHMEG SVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFA WDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKW ERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNF PSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQR LKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKL DITSHNEDYTIVEQYERAEGRHSTGGMDELYK* OT-CD 19-078 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8278 8292 (OT-001303) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG linker (GS); GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5 (Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT, R732L, EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD F736A); linker KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT (GSG); P2A YDALHMQALPPRGSEETRELQSLAAAVVPSAQTLKI linker; mCherry TDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHE (M1L); stop VLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS GLSIEEYKTTLKIIKQAILATDLALYIKRLGEFAELIRK NQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRI AELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ KWQALAEQQGSGGATNFSLLKQAGDVEENPGPLSK GEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEG RPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKA YVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTV TQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMG WEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEV KTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQ YERAEGRHSTGGMDELYK* OT-CD19-100 MALPVTALLLPLALLLHAARPYPYDVPDYADIQMTQ 8279 8293 (OT-001304) TTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT CD8a leader; HA VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQE Tag; CD19 scFV; DIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS CD8a-Tm, (4- GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY 1BB intracellular GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI domain); CD3 zeta IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY signaling domain; AMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSL linker (GS); RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV hPDE5 (Amino LLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED acid 535-860 of GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL WT); linker (GS), YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ Spacer (LD); P2A EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY linker; mCherry QGLSTATKDTYDALHMQALPPRGSEETRELQSLAAA (M1L); stop VVPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNL VQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHA FNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDL DHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLM ILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKR RGEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAI TKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLM NREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPL LDGCRKNRQKWQALAEQQGSLDGATNFSLLKQAGD VEENPGPLSKGEEDNMAIIKEFMRFKVHMEGSVNGH EFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILS PQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVM NFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDG PVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLK DGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSH NEDYTIVEQYERAEGRHSTGGMDELYK* OT-CD 19-101 MALPVTALLLPLALLLHAARPYPYDVPDYADIQMTQ 8280 8294 (OT-001305) TTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT CD 8a leader; HA VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQE Tag; CD19 scFV; DIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS CD8a-Tm; (4- GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY 1BB intracellular GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI domain); CD3 zeta IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY signaling domain; AMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSL Linker (GS); RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV hPDE5(Amino LLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED acid 535-860 of GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL WT, R732L); YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ Linker (GS), EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY Spacer (LD); P2A QGLSTATKDTYDALHMQALPPRGSEETRELQSLAAA linker; mCherry VVPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNL (M1L); stop VQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHA FNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDL DHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLM ILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKR LGEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAI TKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLM NREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPL LDGCRKNRQKWQALAEQQGSLDGATNFSLLKQAGD VEENPGPLSKGEEDNMAIIKEFMRFKVHMEGSVNGH EFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILS PQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVM NFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDG PVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLK DGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSH NEDYTIVEQYERAEGRHSTGGMDELYK* OT-CD 19-111 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8281 8295 (0T-001454) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG Linker (GS); GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5 (Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT, R732L); stop EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRGSEETRELQSLAAAVVPSAQTLKI TDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHE VLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS GLSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRK NQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRI AELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ KWQALAEQQ* OT-CD 19-130 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8282 8296 (OT-001455) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG Linker (GS); GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5 (Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT, H653A); EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD stop KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRGSEETRELQSLAAAVVPSAQTLKI TDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHE VLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA ALKAGKIQNKLTDLEILALLIAALSADLDHRGVNNSY IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS GLSIEEYKTTLKIIKQAILATDLALYIKRRGEFFELIRK NQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRI AELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ KWQALAEQQ* OT-CD 19-131 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8283 8297 (OT-001456) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG Linker (GS); GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5 (Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT, H653A, EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD R732L); stop KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRGSEETRELQSLAAAVVPSAQTLKI TDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHE VLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA ALKAGKIQNKLTDLEILALLIAALSADLDHRGVNNSY IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS GLSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRK NQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRI AELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ KWQALAEQQ* OT-CD 19-132 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLG 8284 8298 (OT-001457) DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR CD8a leader; LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG CD19 scFV; NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ CD8a-Tm; (4- ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR 1BB intracellular KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF domain); CD3 zeta LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG signaling domain; TSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG Linker (GS); GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC hPDE5 (Amino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE acid 535-860 of GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE WT, R732L, EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD D764A); stop KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRGSEETRELQSLAAAVVPSAQTLKI TDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHE VLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFA ALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSY IQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILS GLSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRK NQFNLEDPHQKELFLAMLMTACALSAITKPWPIQQRI AELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPS MQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQ KWQALAEQQ*

Tandem CAR (TanCAR)

In some embodiments, the CAR of the present invention may be a tandem chimeric antigen receptor (TanCAR) which is able to target two, three, four, or more tumor specific antigens. In some aspects, the CAR is a bispecific TanCAR including two targeting domains which recognize two different TSAs on tumor cells. The bispecific CAR may be further defined as comprising an extracellular region comprising a targeting domain (e.g., an antigen recognition domain) specific for a first tumor antigen and a targeting domain (e.g., an antigen recognition domain) specific for a second tumor antigen. In other aspects, the CAR is a multi specific TanCAR that includes three or more targeting domains configured in a tandem arrangement. The space between the targeting domains in the TanCAR may be between about 5 and about 30 amino acids in length, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acids.

Split CAR

In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains of the present invention may be split into two or more parts such that it is dependent on multiple inputs that promote assembly of the intact functional receptor. In one embodiment, the split synthetic CAR system can be constructed in which the assembly of an activated CAR receptor is dependent on the binding of a ligand to the SRE (e.g. a small molecule) and a specific antigen to the targeting moiety. As a non-limiting example, the split CAR consists of two parts that assemble in a small molecule-dependent manner; one part of the receptor features an extracellular antigen binding domain (e.g. scFv) and the other part has the intracellular signaling domains, such as the CD3 intracellular domain.

In other aspects, the split parts of the CAR system can be further modified to increase signal. In one example, the second part of cytoplasmic fragment may be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., CD8a transmembrane domain) to the construct. An additional extracellular domain may also be added to the second part of the CAR system, for instance an extracellular domain that mediates homo-dimerization. These modifications may increase receptor output activity, i.e., T cell activation.

In some aspects, the two parts of the split CAR system contain heterodimerization domains that conditionally interact upon binding of a heterodimerizing small molecule. As such, the receptor components are assembled in the presence of the small molecule, to form an intact system which can then be activated by antigen engagement. Any known heterodimerizing components can be incorporated into a split CAR system. Other small molecule dependent heterodimerization domains may also be used, including, but not limited to, gibberellin-induced dimerization system (GID1-GAI), trimethoprim-SLF induced ecDHFR and FKBP dimerization (Czlapinski et al., J Am Chem Soc., 2008, 130(40): 13186-13187) and ABA (abscisic acid) induced dimerization of PP2C and PYL domains (Cutler et al., Annu Rev Plant Biol. 2010, 61: 651-679). The dual regulation using inducible assembly (e.g., ligand dependent dimerization) and degradation (e.g., destabilizing domain induced CAR degradation) of the split CAR system may provide more flexibility to control the activity of the CAR modified T cells.

Switchable CAR

In some embodiments, the CAR of the invention may be a switchable CAR. Juilerat et al (Juilerat et al., Sci. Rep., 2016, 6: 18950; the contents of which are incorporated herein by reference in their entirety) recently reported controllable CARs that can be transiently switched on in response to a stimulus (e.g. a small molecule). In this CAR design, a system is directly integrated in the hinge domain that separate the scFv domain from the cell membrane domain in the CAR. Such system is possible to split or combine different key functions of a CAR such as activation and co-stimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture. This integrated system can switch the scFv and antigen interaction between on/off states controlled by the absence/presence of the stimulus.

Reversible CAR

In other embodiments, the CAR of the invention may be a reversible CAR system. In this CAR architecture, a LID domain (ligand-induced degradation) is incorporated into the CAR system. The CAR can be temporarily down-regulated by adding a ligand of the LID domain. The combination of LID and DD mediated regulation provides tunable control of continuingly activated CAR T cells, thereby reducing CAR mediated tissue toxicity.

Inhibitory CAR (iCAR)

In some embodiments, payloads of the present invention may be inhibitory CARs. Inhibitory CAR (iCAR) refers to a bispecific CAR design wherein a negative signal is used to enhance the tumor specificity and limit normal tissue toxicity. This design incorporates a second CAR having a surface antigen recognition domain combined with an inhibitory signal domain to limit T cell responsiveness even with concurrent engagement of an activating receptor. This antigen recognition domain is directed towards a normal tissue specific antigen such that the T cell can be activated in the presence of first target protein, but if the second protein that binds to the iCAR is present, the T cell activation is inhibited.

As a non-limiting example, iCARs against Prostate specific membrane antigen (PMSA) based on CTLA4 and PD1 inhibitory domains demonstrated the ability to selectively limit cytokine secretion, cytotoxicity and proliferation induced by T cell activation (Fedorov V. D, et al., 2013, Sci Transl Med, 11; 5(215):215ra172; the contents of which are incorporated herein in their entirety).

Chimeric Switch Receptor

In some embodiments, payloads of the invention may be chimeric switch receptors which can switch a negative signal to a positive signal. As used herein, the term “chimeric switch receptor” refers to a fusion protein comprising a first extracellular domain and a second transmembrane and intracellular domain, wherein the first domain includes a negative signal region and the second domain includes a positive intracellular signaling region. In some aspects, the fusion protein is a chimeric switch receptor that contains the extracellular domain of an inhibitory receptor on T cell fused to the transmembrane and cytoplasmic domain of a co-stimulatory receptor. This chimeric switch receptor may convert a T cell inhibitory signal into a T cell stimulatory signal.

As a non-limiting example, the chimeric switch receptor may comprise the extracellular domain of PD-1 fused to the transmembrane and cytoplasmic domain of CD28 as taught by Liu et al. (Liu et al., Cancer Res., 2016, 76(6): 1578-1590; the contents of which are incorporated by reference in their entirety). In some aspects, extracellular domains of other inhibitory receptors such as CTLA-4, LAG-3, TIM-3, KIRs and BTLA may also be fused to the transmembrane and cytoplasmic domain derived from costimulatory receptors such as CD28, 4-1BB, CD27, OX40, CD40, GTIR and ICOS. In the context of the present invention, the SRE domain (e.g., DD) may be inserted at the N- or C-terminus of the chimeric switch receptor.

In some embodiments, chimeric switch receptors of the present invention may include recombinant receptors comprising the extracellular cytokine-binding domain of an inhibitory cytokine receptor (e.g., IL13 receptor a (IL13Rα1), IL10R, and IL4Rα) fused to an intracellular signaling domain of a stimulatory cytokine receptor such as IL2R (IL2Rα, IL2Rβ and IL2Rgamma) and IL7Ra. One example of such chimeric cytokine receptor is a recombinant receptor containing the cytokine-binding extracellular domain of IL4Ra linked to the intracellular signaling domain of IL7Ra (see, US patent publication NO: 2014/0050709; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, the chimeric switch receptor of the present invention may be a chimeric TGFβ receptor. The chimeric TGFβ receptor may comprise an extracellular domain derived from a TGFβ receptor such as TGFβ receptor 1, TGFβ receptor 2, TGFβ receptor 3, or any other TGFβ receptor or variant thereof; and a non-TGFβ receptor intracellular domain. The non-TGFβ receptor intracellular domain may be the intracellular domain or fragment thereof derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CD28, 4-1BB (CD137), OX40 (CD134), CD3zeta, CD40, CD27, or a combination thereof. One example of such chimeric TGFβ receptor is discussed in US patent publication NO.: US2016/0075755; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, payloads of the present invention may be bipartite fusion receptors. In one aspect, the bipartite fusion receptor may comprise an antigen-binding domain that binds to a tumor antigen and an activation domain that binds to one or more cell surface molecules. In some aspects, the antigen binding domain of a bipartite receptor is a scFv antibody. In other aspects, the activation domain that binds to a T cell surface stimulatory molecule may be selected from CD3, CD27, CD 28, CD40, OX40 (CD134) and 4-1BB (CD137), or a NK cell surface stimulatory molecule selected from CD16, NKG2D and NKp30. The activation domain may also be a scFv specific to a stimulatory molecule on the surface of immune cells (e.g., T cells and NK cells). Immune cells can be genetically modified to express a bipartite molecule comprising at least one antigen binding domain and an activation domain. The antigen binding domain binds to one or more molecules present on target cells such as cancer cells. Immune cells that express the molecule recognized by the activation domain will be activated and attack the recognized cancer cells. As a non-limiting example, a bipartite molecule may be an engager molecule comprising an antigen recognition scFv specific to CD19 or EphA2, as described in US patent publication NO: US2016/0015749; the contents of which are incorporated by reference herein in their entirety.

Activation-Conditional CAR

In some embodiments, payloads of the invention may be an activation-conditional chimeric antigen receptor, which is only expressed in an activated immune cell. The expression of the CAR may be coupled to activation conditional control region which refers to one or more nucleic acid sequences that induce the transcription and/or expression of a sequence e.g. a CAR under its control. Such activation conditional control regions may be promoters of genes that are upregulated during the activation of the immune effector cell e.g. IL2 promoter or NFAT binding sites. In some embodiments, activation of the immune cell may be achieved by a constitutively expressed CAR (International Publication No: WO 2016126608; the contents of which are incorporated herein by reference in their entirety).

CAR Targeting to Tumor Cells with Specific Proteoglycan Markers

In some embodiments, payloads of the present invention may be a CAR that targets specific types of cancer cells. Human cancer cells and metastasis may express unique and otherwise abnormal proteoglycans, such as polysaccharide chains (e.g., chondroitin sulfate (CS), dermatan sulfate (DS or CSB), heparan sulfate (HS) and heparin). Accordingly, the CAR may be fused with a binding moiety that recognizes cancer associated proteoglycans. In one example, a CAR may be fused with VAR2CSA polypeptide (VAR2-CAR) that binds with high affinity to a specific type of chondroitin sulfate A (CSA) attached to proteoglycans. The extracellular ScFv portion of the CAR may be substituted with VAR2CSA variants comprising at least the minimal CSA binding domain, generating CARs specific to chondroitin sulfate A (CSA) modifications. Alternatively, the CAR may be fused with a split-protein binding system to generate a spy-CAR, in which the scFv portion of the CAR is substituted with one portion of a split-protein binding system such as SpyTag and Spy-catcher and the cancer-recognition molecules (e.g. scFv and or

VAR2-CSA) are Attached to the CAR Through the Split-Protein Binding System (See, e.g., PCT publication No.: WO2016/135291; the contents of which are incorporated by reference in their entirety.) SUPRA CAR

In some embodiments, the payload of the present invention may be a Split Universal Programmable (SUPRA) CAR. A SUPRA CAR may be a two-component receptor system comprising of a universal receptor (zip CAR) expressed on T cells and a tumor-targeting scFv adaptor. The zip CAR universal receptor may be generated by the fusion of intracellular signaling domains and a leucine zipper as the extracellular domain. The tumor-targeting scFv adaptor molecule or zipFv, may be generated by the fusion of a cognate leucine zipper and an scFv. The scFv of the zipFv may bind to a tumor antigen, and the leucine zipper may bind and activate the zip CAR on the T cells. Unlike the conventional fixed CAR design, the SUPRA CAR modular design allows targeting of multiple antigens without further genetic manipulations of the immune cells. Any of the CAR designs disclosed by Cho et al., 2018, Cell 173, 1-13, may be useful in the present invention (the contents of which are incorporated by reference in their entirety).

6. Cytokines, Chemokines and Other Soluble Factors

In accordance with the present invention, payloads of the present invention may be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells and other cell types, which act as chemical communicators between cells and tissues within the body. These proteins mediate a wide range of physiological functions, from effects on cell growth, differentiation, migration and survival, to a number of effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic function to eliminate tumor cells.

In some embodiments, payloads of the present invention may be cytokines, and fragments, variants, analogs and derivatives thereof, including but not limited to interleukins, tumor necrosis factors (TNFs), interferons (IFNs), TGF beta and chemokines. In some embodiments, payloads of the present invention may be cytokines that stimulate immune responses. In other embodiments, payloads of the invention may be antagonists of cytokines that negatively impact anti-cancer immune responses.

In some embodiments, payloads of the present invention may be cytokine receptors, recombinant receptors, variants, analogs and derivatives thereof; or signal components of cytokines.

In some embodiments, cytokines of the present invention may be utilized to improve expansion, survival, persistence, and potency of immune cells such as CD8+T_(EM), natural killer cells and tumor infiltrating lymphocytes (TIL) cells used for immunotherapy. In other embodiments, T cells engineered with two or more DD regulated cytokines are utilized to provide kinetic control of T cell activation and tumor microenvironment remodeling. In one aspect, the present invention provides biocircuits and compositions to minimize toxicity related to cytokine therapy. Despite its success in mitigating tumor burden, systemic cytokine therapy often results in the development of severe dose limiting side effects. Two factors contribute to the observed toxicity (a) Pleiotropism, wherein cytokines affect different cells types and sometimes produce opposing effects on the same cells depending on the context (b) Cytokines have short serum half-life and thus need to be administered at high doses to achieve therapeutic effects, which exacerbates the pleiotropic effects. In one aspect, cytokines of the present invention may be utilized to modulate cytokine expression in the event of adverse effects. In some embodiments, cytokines of the present invention may be designed to have prolonged life span or enhanced specificity to minimize toxicity.

In some embodiments, the payload of the present invention may be an interleukin (IL) cytokine. Interleukins (ILs) are a class of glycoproteins produced by leukocytes for regulating immune responses. As used herein, the term “interleukin (IL)” refers to an interleukin polypeptide from any species or source and includes the full-length protein as well as fragments or portions of the protein. In some aspects, the interleukin payload is selected from IL1, IL1alpha (also called hematopoietin-1), IL1beta (catabolin), IL1 delta, IL1epsilon, IL1eta, IL1 zeta, interleukin-1 family member 1 to 11 (IL1F1 to IL1F11), interleukin-1 homolog 1 to 4 (IL1H1 to IL1H4), IL1 related protein 1 to 3 (IL1RP1 to IL1RP3), IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL10C, IL10D, IL11, IL11a, IL11b, IL12, IL13, IL14, IL15, IL16, IL17, IL17A, IL17B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20 like (IL20L), IL21, IL22, IL23, IL23A, IL23-p19, IL23-p40, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36 alpha, IL36 beta, IL36 gamma, IL36RN, IL37, IL37a, IL37b, IL37c, IL37d, IL37e and IL38. In other aspects, the payload of the present invention may be an interleukin receptor selected from CD121a, CDw121b, IL2Rα/CD25, IL2Rβ/CD122, IL2Rγ/CD132, CDw131, CD124, CD131, CDw125, CD126, CD130, CD127, CDw210, IL8RA, IL11Rα, CD212, CD213α1, CD213α2, IL14R, IL15Rα, CDw217, IL18Rα, IL18Rβ, IL20Rα, and IL20Rβ.

In certain embodiments, a cytokine may be a type I interferons (IFN) including IFN alpha subtypes (IFN α1, IFN α1b, IFN α1c), IFN beta, IFN delta subtypes (IFN delta 1, IFN delta 2, IFN delta 8), IFN gamma, IFN kappa, and IFN epsilon, IFN lambda, IFN omega, IFN tau and IFN zeta. In certain embodiments, a cytokine may be a member of tumor necrosis factor (TNF) superfamily, including TNF-alpha, TNF-beta (also known as lymphotoxin-alpha (LT-α)), lymphotoxin-beta (LT-β), CD40L (CD154), CD27L (CD70), CD30L (CD153), FASL (CD178), 4-1BBL (CD137L), OX40L, TRAIL (TNF-related apoptosis inducing ligand), APRIL (a proliferation-inducing ligand), TWEAK, TRANCE, TALL-1, GITRL, LIGHT and TNFSF1 to TNFSF20 (TNF ligand superfamily member 1 to 20).

In one embodiment, the payload of the invention may comprise IL2 (SEQ ID NO. 8299, encoded by SEQ ID NO. 8300 and 8301). In one aspect, the effector module of the invention may be a DD-IL2 fusion polypeptide.

In some aspects of the invention, an IL2 mutein may be used as a payload. As used herein, the term “mutein” is a construct, molecule or sequence of a mutation, change or alteration in a protein and hence is also known as a mutant, e.g., a protein mutant, mutein. Consequently, an “IL2 mutein” is an IL2 mutant. In some embodiments an IL2 mutein is a variant of wild type IL2 protein, where the wildtype IL2 consists of the amino acid sequence of SEQ ID NO. 8299. In some aspects, it refers to an IL2 variant which binds to and activates only cells expressing IL2Rαβγ, but does not significantly bind to or activate cell expressing only IL2Rβγ. In some examples, an IL2 mutein may be an IL2 protein in which residues of IL2 responsible for binding to either IL2Rβ or IL2Rγ are substituted and abolish their interaction. In other examples, an IL2 mutein may be an IL2 protein comprising mutations conferring high affinity for IL2Rα. An IL2 mutein may be an IL2 selective agonist (IL2_(SA)) which can preferentially activate the high affinity IL2 receptor (i.e., IL2Rαβγ) which is necessary to selectively activate T cells with respect to NK cells. In some embodiments, the IL2 mutein may be IL2 protein which preferentially binds to the lower affinity IL2Rβγ but with reduced affinity to CD25.

In some embodiments, IL2 muteins may be used to preferentially expand or stimulate Treg cells. As used herein “preferentially expand or stimulate Treg cells” means the IL2 muteins promote the proliferation, survival, activation and/or function of T regulatory cells.

Exemplary IL2 muteins may include, but are not limited to, N88R substitution (Shanafelt et al., Nature Biotech., 2000, 18:1197-1202), an IL2 with a V91K substitution (e.g., US Patent publication NO. US20140286898); V91K substitution, C125A substitution, an IL2 with three mutations: V69A, N71R, Q74P; an IL2 mutein with high affinity for IL2Rα (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P); an IL2 mutein with high affinity for IL2Ra and reduced signaling activity (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D), and D20H, D20I, N88G, N88I, N88R, and Q126L substitutions as described in PCT application NO. 1999060128; the contents of each of which are incorporated herein by reference in their entirety. In other aspects, IL2 muteins may include those described in U.S. Pat. Nos. 4,518,584; 5,116,943; 5,206,344; 6,955,807; 7,105,653; 7,371,371; 7,803,361; 8,124,066; 8,349,311; 8,759,486; and 9,206,243; PCT patent publication NOs: WO2005086751 and WO2012088446; EP Pat. NOs: EP0234599 and EP0200280 and Sim, G. C. et al. (2016) Cancer Immunol Res; 4(11):983-994; the contents of each of which are incorporated herein by reference in their entirety.

In some aspects, the IL2 mutein may be fused to a polypeptide that extends the serum half-life of the IL2 mutein, such as an IgG Fc fragment. Preferred Fc regions are derived from human IgG, which includes IgG1, IgG2, IgG3, and IgG4. In other aspects, the payload of the invention may be an IL2 fusion protein comparing a second functional polypeptide. In a non-limiting example, an IL2 fusion protein may comprise an IL2 or IL2 mutein polypeptide fused with a pro-apoptotic Bcl-2 family polypeptide (such as Bad, Bik/Nbk, Bid, Bim/Bod, Hrk, Bak or Bax); such fusion protein may be capable of inhibiting cell survival, inhibiting cell proliferation, or enhancing cell death or apoptosis of a target cell expressing an IL2 receptor. Alternatively, an IL2 or IL2 mutein polypeptide may be fused with an anti-apoptotic Bc1-2 family polypeptide (such as Bc1-x_(L), Bc1-w or Bc1-2). The fusion protein may be capable of enhancing cell survival, enhancing cell proliferation, or inhibiting cell death or apoptosis of a target cell expressing an IL2 receptor. See, e.g., US patent publication NO.: US2016/0229901.

In addition, the IL2 fusion protein may be a IL2-GMCSF fusion protein which can promote cell-cell interaction; therefore, enhances anti-cancer immune responses (Wen et al., J. Translational Med., 2016, 14: 41).

In one embodiment, the payload of the invention may comprise IL12. IL12 is a heterodimeric protein of two subunits (p35, p40) that is secreted by antigen presenting cells, such as macrophages and dendritic cells. IL12 is type 1 cytokine that acts on natural killer (NK) cells, macrophages, CD8⁺ Cytotoxic T cells, and CD4⁺ T helper cells through STAT4 pathway to induce IFN γ production in these effector immune cells (reviewed by Trinchieri G, Nat Rev Immunol. 2003; 3(2): 133-146). IL12 can promote the cytotoxic activity of NK cells and CD8⁺ T cells, therefore has anti-tumor function. Intravenous injection of recombinant IL12 exhibited modest clinical efficacy in a handful of patients with advanced melanoma and renal cell carcinoma (Gollob et al., Clin. Cancer Res. 2000; 6(5):1678-1692). IL12 has been used as an adjuvant to enhance cytotoxic immunity using a melanoma antigen vaccine, or using peptide pulsed peripheral blood mononuclear cells; and to promote NK cell activity in breast cancer with trastuzumab treatment. Local delivery of IL12 to the tumor microenvironment promotes tumor regression in several tumor models. These studies all indicate that locally increased IL12 level can promote anti-tumor immunity. One major obstacle of systemic or local administration of recombinant IL12 protein, or through oncolytic viral vectors is the severe side effects when IL12 is presented at high level. Developing a system that tightly controls IL12 level may provide a safe use of IL12 in cancer treatment.

In one aspect, the effector module of the invention may be a DD-IL12 fusion polypeptide. This regulatable DD-IL12 fusion polypeptide may be directly used as an immunotherapeutic agent or be transduced into an immune effector cell (T cells and TIL cells) to generate modified T cells with greater in vivo expansion and survival capabilities for adoptive cell transfer. The need for harsh preconditioning regimens in current adoptive cell therapies may be minimized using regulated IL12; DD-IL12 may be utilized to modify tumor microenvironment and increase persistence in solid tumors that are currently refractory to tumor antigen targeted therapy. In some embodiments, CAR expressing T cells may be armored with DD regulated IL12 to relieve immunosuppression without systemic toxicity.

In some embodiments, the IL12 may be a Flexi IL12, wherein both p35 and p40 subunits, are encoded by a single cDNA that produces a single chain polypeptide. In one embodiment, the IL12 may comprise p40 subunit, which includes amino acids 23-328 of wildtype IL12B and comprise the amino acid sequence of SEQ ID NO. 8302 (encoded by SEQ ID NO. 8303-8312) and a p35 subunit, which includes amino acids 57-253 of wildtype IL12A and comprise the amino acid sequence of SEQ ID NO. 8313 (encoded by SEQ ID NO. 8314-8323). Any portion of IL12 that retains one or more functions of full length or mature IL12 may be useful in the present invention.

In some embodiments, DD regulated IL12 compositions of the invention may be utilized to minimize the cytotoxicity associated with systemic IL12 administration. Treatment with IL12 has been associated with systemic flu-like symptoms (fever, chills, fatigue, arthromyalgia, headache), toxic effects on the bone marrow, and liver. Hematologic toxicity observed most commonly included neutropenia and thrombocytopenia; hepatic dysfunction manifested in transient (dose dependent) increase in transaminases, hyperbilirubinemia and hypoalbuminemia. In some instances, toxicity is also associated with inflammation of the mucus membranes (oral mucositis, stomatitis or colitis). These toxic effects of IL12 were related to the secondary production of IFNγ, TNF alpha, and chemokines such as IP10, and MIG. In certain aspects of the invention, DD regulated IL12 may be utilized to prevent the toxic effects associated with elevated production of secondary messengers. In some embodiments, DD regulated Flexi-IL12 constructs may be used to improve the efficacy of the CARs, especially in solid tumor settings, by providing a controlled local signal for tumor microenvironment remodeling and epitope spreading. DD regulation also provides rapid, dose dependent, and local production of Flexi IL12.

The format of the IL12 constructs utilized as payload of the present invention may be optimized. In one embodiment, the payload of the invention may be a bicistronic IL12 containing p40 and p35 subunits separated by an internal ribosome entry site or a cleavage site such as P2A or Furin to allow independent expression of both subunits from a single vector. This results in a configuration of secreted IL12 that is more akin to the naturally occurring IL12 than the flexi IL12 construct, the payload of the invention may be the p40 subunit of the IL12. DD regulated p40 may be co-expressed with constitutive p35 construct to generate “regulatable IL12” expression. Alternatively, the DD regulated p40 may heterodimerize with the endogenous p35. p40 has been shown to stabilize p35 expression and stimulate the export of p35 (Jalah R, et al. (2013). Journal of Biol. Chem. 288, 6763-6776 (the contents of which are incorporated by reference in its entirety).

In some embodiments, modified forms of IL12 may be utilized as the payload. These modified forms of IL12 may be engineered to have shortened half-life in vivo compared to the non-modified form of especially when used in combination with tunable systems described herein.

Human flexi IL12 has a reported half-life of 5-19 hours which, when administered as a therapeutic compound, can result in systemic cytotoxicity (Car et al. (1999) The Toxicology of Interleukin-12: A Review” Toxicologic Path.27 A, 58-63; Robertson et al. (1999) “Immunological Effects of Interleukin 12 Administered by Bolus Intravenous Injection to Patients with Cancer” Clin. Cancer Res. 5:9-16; Atkins et al. (1997)“Phase I Evaluation of Intravenous Recombinant Human Interleukin 12 in Patients with Advance Malignancies” Clin. Cancer Res. 3:409-417). The ligand inducible control of IL12 can regulate production in a dose dependent fashion, the time from cessation of ligand dosing to cessation of protein synthesis and IL12 clearance may be insufficient to prevent toxic accumulation of IL12 in plasma.

In one embodiment, the modified form of IL12 utilized as the payload may be a Topo-sc IL12 which have the configuration as follows from N to C terminus (i) a first IL12 p40 domain (p40N), (ii) an optional first peptide linker, (iii) an IL12 p35 domain, (iv) an optional second peptide linker, and (v) a second IL12 p40 domain (p40C). In one embodiment, modified topo sc IL12 polypeptides exhibit increased susceptibility to proteolysis. Topo-sc IL12 is described in International Patent Publication No. WO2016048903; the contents of which are incorporated herein by reference in its entirety.

IL12 polypeptide may also be modified (e.g. genetically, synthetically, or recombinantly engineered) to increase susceptibility to proteinases to reduce the biologically active half-life of the IL12 complex, compared to a corresponding IL12 lacking proteinases susceptibility. Proteinase susceptible forms of IL12 are described in International Patent Publication No. WO2017062953; the contents of which are incorporated by reference in its entirety.

IL12 systemic toxicity may also be limited or tightly controlled via mechanisms involving tethering IL12 to the cell surface to limit its therapeutic efficacy to the tumor site. Membrane tethered IL12 forms have been described previously using Glycosyl phosphatidylinositol (GPI) signal peptide or using CD80 transmembrane domain (Nagarajan S, et al. (2011) J Biomed Mater Res A. 99(3):410-7; Bozeman E N, et al. (2013) Vaccine. 7; 31(20):2449-56; Wen-Yu Pan et al. (2012), Mol. Ther. 20:5, 927-937; the contents of each of which are incorporated by reference in their entirety). In some embodiments, transmembrane domains may be selected from any of those described in Table 13, Table 14, and Table 15.

In some embodiments, the IL12 levels secreted by the immune cells of the invention may approximately be comparable to the IL12 levels secreted by human myeloid dendritic cells (mDC1), when activated with TLR agonists. In one embodiment, the TLR agonist may be the combination of lipopolysaccharide administered with R848.

In some embodiments, the IFN gamma secreted by IL12 induced activation of the immune cells is at least 5-fold greater in the presence of ligand, compared to the levels in the absence of ligand.

In some embodiments, regulation of IL12 provides the necessary safety switch. In some embodiments, IL-12 secretion recruit and/or activates effector cells in the tumor microenvironment. In some embodiments, the IL12 regulation provides a benefit to CAR T function without causing toxicity.

In one embodiment, the payload of the invention may comprise IL15. Interleukin 15 is a potent immune stimulatory cytokine and an essential survival factor for T cells, and Natural Killer cells. Preclinical studies comparing IL2 and IL15, have shown than IL15 is associated with less toxicity than IL2. In some embodiments, the effector module of the invention may be a DD-IL15 fusion polypeptide. IL15 polypeptide may also be modified to increase its binding affinity for the IL15 receptor. For example, the asparagine may be replaced by aspartic acid at position 72 of IL15 (SEQ ID NO. 2 of US patent publication US20140134128A1; the contents of which are incorporated by reference in their entirety). In some aspects, the IL15 comprises amino acid sequence of SEQ ID NO. 8234 (encoded by SEQ ID NO. 8236), which include amino acids 49-162 of wildtype IL15. In some embodiments the IL15 sequence may include a stop codon and may be encoded by the nucleotide sequence of SEQ ID NO. 8235, and 8237-8238.

A unique feature of IL15 mediated activation is the mechanism of trans-presentation in which IL15 is presented as a complex with the alpha subunit of IL15 receptor (IL15Rα) that binds to and activates membrane bound IL15 beta/gamma receptor, either on the same cell or a different cell. The IL15/IL15Ra complex is more effective in activating IL15 signaling, than IL15 by itself. Thus, in some embodiments, the effector module of the invention may include a DD-IL15/IL15Ra fusion polypeptide. In one embodiment, the payload may be IL15/IL15Ra fusion polypeptide described in US Patent Publication NO.: US20160158285A1 (the contents of which are incorporated herein by reference in their entirety). The IL15 receptor alpha comprises an extracellular domain called the sushi domain which contains most of the structural elements necessary for binding to IL15. Thus, in some embodiments, payload may be the IL15/IL15Ra sushi domain fusion polypeptide described in US Patent Publication NO.: US20090238791A1 (the contents of which are incorporated herein by reference in their entirety).

Regulated IL15/IL15Ra may be used to promote expansion, survival and potency of CD8T_(EM) cell populations without impacting regulatory T cells, NK cells and TIL cells. In one embodiment, DD-IL15/IL15Ra may be utilized to enhance CD19 directed T cell therapies in B cell leukemia and lymphomas. In one aspect, IL15/IL15Ra may be used as payload of the invention to reduce the need for pre-conditioning regimens in current CAR-T treatment paradigms.

The effector modules containing DD-IL15, DD-IL15/IL15Ra and/or DD-IL15/IL15Ra sushi domain may be designed to be secreted (using e.g. IL2 signal sequence) or membrane bound (using e.g. IgE or CD8a signal sequence).

In some embodiments, the IFN gamma secreted by IL15 induced activation of the immune cells is at least 10-fold greater in the presence of ligand, compared to the levels in the absence of ligand.

In some embodiments, regulation of IL15-IL15Ra fusion proteins provides a safety switch as compared to constitutively expressed IL15-IL15Rα. In some embodiments, IL15 -IL15Ra leads to better expansion, and/or persistence of CAR T cells.

In some aspects, the DD-IL115/IL15Ra comprises the amino acid sequences provided in Table 18. In some embodiments, the linker utilized in Table 18 may be SG linker. In Table 18, asterisk indicates the translation of the stop codon. In some embodiments, the DDs described in Table 18 may contain an additional stop codon. As used herein the wildtype (WT) of IL15 refers to Uniprot ID: P40933 and wildtype (WT) of IL15Ra refers to UniProt ID: Q13261. Table 18 also provides alternate aliases for a given construct ID. These aliases are identified by the prefix OT.

TABLE 18 DD-IL15-IL15Ra construct sequences AA NA Description/ SEQ ID SEQ ID Construct ID Amino Acid Sequence NO NO IL15 (49-162 of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTA 8324 8326; WT) MKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNV 8344 TESGCKECEELEEKNIKEFLQSFVHIVQMFINTS IL15Ra (31-267 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 8329 8345; of WT) SLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPST 8346 VTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGS QLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASAS HQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKS RQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL IgE leader MDWTWILFLVAAATRVHS  276  277 SG3-(SG4)3- SGGGSGGGGSGGGGSGGGGSGGGSLQ  323  324 SG3-SLQ linker SG Linker SG — AGTG GT GSGSGS linker GSGSGS 8330 8347 GSGSGSGS GSGSGSGS 8331 8348 linker GSGSGGGSGS GSGSGGGSGS 8332 8349 linker hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCT    3  339 acid 535-860 of IRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH WT) NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKP WPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCT   12  359 acid 535-860 of IRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH WT, R732L) NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRL GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKP WPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQ hPDE5 (Amino EETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCT  227  233 acid 535-860 of IRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH WT, R732L, NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS F736A) HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRL GEFAELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKP WPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQ AcGFP (Amino VSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYG   79  372 acid 2-239 of KLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK WT) QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDT LVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDK AKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLP DNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDE LYK FLAG-tag DYKDDDDK 8333 8350 HA Tag YPYDVPDYA 8238 8351 Modified Furin ESRRVRRNKRSK  288  291 OT-IL15-031 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHI 8334 8352 (OT-001254, DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD OT-IL15-045) TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS IgE signal FVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQI sequence; IL15 TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS (Amino acid LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTV 49-162 of WT); TTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQ linker (SG3- LMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH (SG4)3-SG3- QPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSR SLQ); IL15Ra QTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLSGE (Amino acid ETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI 31-267 of WT); RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH linker (SG); NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS hPDE5 (Amino HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL acid 535-860 of MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRL WT, R732L, GEFAELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKP F736A); stop WPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK TGA NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQ* OT-IL15-032 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHI 8335 8353 (OT-001469) DATLYlESDVHPSCKVTAMKCFLLELQVISLESGDASIHD IgE signal TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS sequence; IL15 FVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQI (Amino acid TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS 49-162 of WT); LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTV linker (SG3- TTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQ (SG4)3-SG3- LMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH SLQ); IL15Ra QPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSR (Amino acid QTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLSG 31-267 of WT); VSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYG linker (SG); KLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMK AcGFP (Amino QHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDT acid 2-239 of LVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDK WT); linker AKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLP (SG); hPDE5 DNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDE (Amino acid LYKSGEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDL 535-860 of WT, ETALCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYR R732L, KNVAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILA F736A); stop LLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHH HFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILATDL ALYIKRLGEFAELIRKNQFNLEDPHQKELFLAMLMTACD LSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDL MNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLD GCRKNRQKWQALAEQQ* OT-IL15-033 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHI 8336 8354 (OT-001470) DATLYIESDVHPSCKVTAMKCFLLELQVISLESGDASIHD IgE signal TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS sequence; IL15 FVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQI (Amino acid TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS 49-162 of WT); LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTV linker (SG3- TTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQ (SG4)3-SG3- LMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH SLQ); IL15Ra QPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSR (Amino acid QTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLSGE 31-267 of WT); ETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI linker (SG); RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH hPDE5 (Amino NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS acid 535-860 of HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, R732L, MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRL F736A); linker GEFAELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKP (SG); AcGFP WPIQQRIAELVAIEFFDQGDRERKELNIEPTDLMNREKK (Amino acid 2- NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR 239 of WT); QKWQALAEQQSGVSKGAELFTGIVPILIELNGDVNGHKF stop SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSY GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGN YKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYN YNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF GFVTAAAITHGMDELYK* OT-IL15-043 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHI 8337 8355 (OT-001315) DATLYIESDVHPSCKVTAMKCFLLELQVISLESGDASIHD IgE signal TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS sequence; IL15 FVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQI (Amino acid TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS 49-162 of WT); LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTV Linker (SG3- TTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQ (SG4)3-SG3- LMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH SLQ); IL15Ra  QPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSR (Amino acid QTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLSGE 31-267 of WT); SRRVRRNKRSKEETRELQSLAAAVVPSAQTLKITDFSFSD Linker (SG); FELSDLETALCTIRMFTDLNLVQNFQMKHEVLCRWILSV Furin cleavage KKNYRKNVAYHNWRHAFNTAQCMFAALKAGKIQNKLT site DLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHS (ESRRVRRNK IMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAIL RSK); hPDE5 ATDLALYIKRLGEFFELIRKNQFNLEDPHQKELFLAMLM (Amino acid TACDLSAITKPWPIQQRIAELVAIEFFDQGDRERKELNIEP 535-860 of WT, TDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFP R732L); stop LLDGCRKNRQKWQALAEQQ* OT-IL15-044 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHI 8338 8356 (OT-001316) DATLYIESDVHPSCKVTAMKCFLLELQVISLESGDASIHD IgE signal TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS sequence; IL15 FVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQI (Amino acid TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS 49-162 of WT); LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTV Linker (SG3- TTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQ (SG4)3-SG3- LMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH SLQ); IL15Ra QPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSR (Amino acid QTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLSGE 31-267 of WT); ETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI Linker (SG); RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH hPDE5 (Amino NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS acid 535-860 of HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT, R732L); MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRL stop GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKP WPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQ* OT-IL15-048 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHI 8339 8357 (OT-001317) DATLYlESDVHPSCKVTAMKCFLLELQVISLESGDASIHD IgE signal TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS sequence; IL15 FVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQI (Amino acid TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS 49-162 of WT); LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTV Linker (SG3- TTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQ (SG4)3-SG3- LMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH SLQ); IL15Ra QPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSR (Amino acid QTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLSGE 31-267 of WT); ETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI linker (SG); RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH hPDE5 (Amino NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS acid 535-860 of HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT); stop MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKP WPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQ* OT-IL15-194 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHI 8340 8358 (OT-001499) DATLYIESDVHPSCKVTAMKCFLLELQVISLESGDASIHD IgE signal TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS sequence; IL15 FVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQI (Amino acid TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS 49-162 of WT); LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTV Linker (SG3- TTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQ (SG4)3-SG3- LMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH SLQ); IL15Ra QPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSR (Amino acid QTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLSGE 31-267 of WT); ETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTI linker (SG); RMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYH hPDE5 (Amino NVVRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALS acid 535-860 of HDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL WT); stop MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRR GEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKP WPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKK NKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQ* OT-IL15-111 MDWTWILFLVAAATRVHSDYKDDDDKNWVNVISDLKK 8341 8359 (OT-001344) IEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS IgE signal LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELE sequence; Flag EKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGG tag; IL15 GGSGGGSLQYPYDVPDYAITCPPPMSVEHADIWVKSYSL (Amino acid YSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPS 49-162 of WT); LKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPA Linker (SG3- ASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESS (SG4)3-SG3- HGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAIST SLQ); HA tag; STVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPV IL15Ra (Amino TWGTSSRDEDLENCSHHLGSGSGSEETRELQSLAAAVVP acid 31-267 of SAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQ WT); linker MKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMF (GSGSGS); AALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQ hPDE5 (Amino RSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIE acid 535-860 of EYKTTLKIIKQAILATDLALYIKRLGEFFELIRKNQFNLED WT, R732L); PHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFD stop TGA QGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQL YEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ* OT-IL 15-112 MDWTWILFLVAAATRVHSDYKDDDDKNWVNVISDLKK 8342 8360 (OT-001345) IEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS IgE signal LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELE sequence; Flag EKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGG tag; IL15 GGSGGGSLQYPYDVPDYAITCPPPMSVEHADIWVKSYSL (Amino acid YSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPS 49-162 of WT); LKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPA Linker (SG3- ASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESS (SG4)3-SG3- HGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAIST SLQ); HA tag; STVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPV IL15Ra (Amino TWGTSSRDEDLENCSHHLGSGSGSGSEETRELQSLAAAV acid 31-267 of VPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNF WT); linker QMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQC (GSGSGSGS); MFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNS hPDE5 (Amino YIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSG acid 535-860 of LSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRKNQFN WT, R732L); LEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATE stop FFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAIC LQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ* OT-IL 15-113 MDWTWILFLVAAATRVHSDYKDDDDKNWVNVISDLKK 8343 8361 (OT-001346) IEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS IgE signal LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELE sequence; Flag EKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGG tag; IL15 GGSGGGSLQYPYDVPDYAITCPPPMSVEHADIWVKSYSL (Amino acid YSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPS 49-162 of WT); LKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPA Linker (SG3- ASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESS (SG4)3-SG3- HGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAIST SLQ); HA tag; STVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPV IL15Ra (Amino TWGTSSRDEDLENCSHHLGSGSGGGSGSEETRELQSLAA acid 31-267 of AVVPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLV WT); linker QNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTA (GSGSGGGSGS); QCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVN hPDE5 NSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQIL (Amino acid SGLSIEEYKTTLKIIKQAILATDLALYIKRLGEFFELIRKNQ 535-860 of WT, FNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELV R732L); stop ATEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFID AICLQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQ*

In some embodiments, the sequences described in Table 18, may contain an additional stop codon. For example, the construct “hPDE5 (Amino acid 535-860 of WT, R732L); stop” may be encoded by the nucleotide sequence of SEQ ID NO. 8362, and the construct “hPDE5 (Amino acid 535-860 of WT, R732L, F736A); stop” may be encoded by the nucleotide sequence of SEQ ID NO. 8363.

In one embodiment, the payload of the present invention may comprise IL18. IL18 is a proinflammatory and immune regulatory cytokine that promotes IFNγ production by T and NK cells. IL18 belongs to the IL1 family. Secreted IL18 binds to a heterodimer receptor complex, consisting of IL18Rα and β-chains and initiates signal transduction. IL18 acts in concert with other cytokines to modulate immune system functions, including induction of IFN γ production, Th1 responses, and NK cell activation in response to pathogen products. IL18 showed anti-cancer effects in several tumors. Administration of recombinant IL18 protein or IL18 transgene induces melanoma or sarcoma regression through the activation of CD4⁺ T and/or NK cell-mediated responses (reviewed by Srivastava et al., Curr. Med. Chem., 2010, 17: 3353-3357). The combination of IL18 with other cytokines, such as IL12 or co-stimulatory molecules (e.g., CD80) increases IL18 anti-tumor effects. For example, IL18 and IL12A/B or CD80 genes have been integrated successfully in the genome of oncolytic viruses, with the aim to trigger synergistically T cell-mediated anti-tumor immune responses (Choi et al., Gene Ther., 2011, 18: 898-909). IL2/IL18 fusion proteins also display enhanced anti-tumor properties relative to either cytokine alone and low toxicity in preclinical models (Acres et al., Cancer Res., 2005, 65:9536-9546).

IL18 alone, or in combination of IL12 and IL15, activates NK cells. Preclinical studies have demonstrated that adoptively transferred IL12, IL15 and IL18 pre-activated NK cells display enhanced effector function against established tumors in vivo (Ni et al., J Exp Med. 2012, 209: 2351-2365; and Romee et al., Blood. 2012, 120:4751-4760). Human IL12/IL15/IL18 activated NK cells also display memory-like features and secrete more IFN γ in response to cytokines (e.g., low concentration of IL2). In one embodiment, the effector module of the present invention may be a DD-IL18 fusion polypeptide.

In one embodiment, the payload of the present invention may comprise IL21. IL21 is another pleiotropic type I cytokine that is produced mainly by T cells and natural killer T (NKT) cells. IL21 has diverse effects on a variety of cell types including but not limited to CD4⁺ and CD8⁺ T cells, B cells, macrophages, monocytes, and dendritic cells (DCs). The functional receptor for IL21 is composed of IL21 receptor (IL21R) and the common cytokine receptor gamma chain, which is also a subunit of the receptors for IL2, IL4, IL7, IL9 and IL15. Studies provide compelling evidence that IL21 is a promising immunotherapeutic agent for cancer immunotherapy. IL21 promotes maturation, enhances cytotoxicity, and induces production of IFN γ and perforin by NK cells. These effector functions inhibit the growth of B16 melanoma (Kasaian et al., Immunity. 2002, 16(4):559-569; and Brady et al., J Immunol. 2004, 172(4):2048-2058). IL21 together with IL15 expands antigen-specific CD8⁺ T-cell numbers and their effector function, resulting in tumor regression (Zeng et al., J Exp Med. 2005, 201(1):139-148). IL21 may also be used to rejuvenate multiple immune effector cells in the tumor microenvironment. IL21 may also directly induce apoptosis in certain types of lymphoma such as diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia cells, via activation of STAT3 or STAT1 signal pathway. IL21, alone or in combination with anti-CD20 mAb (rituximab) can activate NK cell-dependent cytotoxic effects. Interestingly, discovery of the immunosuppressive actions of IL21 suggests that this cytokine is a “double-edged sword”-IL21 stimulation may lead to either the induction or suppression of immune responses. Both stimulatory and suppressive effects of IL21 must be considered when using IL21-related immunotherapeutic agents. The level of IL21 needs to be tightly controlled by regulatory elements. In one aspect, the effector module of the present invention may be a DD-IL21 fusion polypeptide.

In some embodiments, payloads of the present invention may comprise type I interferons. Type I interferons (IFNs-I) are soluble proteins important for fighting viral infection in humans. IFNs-I include IFN alpha subtypes (IFN α1, IFN α1b, IFN α1c), IFN beta, IFN delta subtypes (IFN delta 1, IFN delta 2, IFN delta 8), IFN gamma, IFN kappa, and IFN epsilon, IFN lambda, IFN omega, IFN tau and IFN zeta. IFN α and IFN β are the main IFN I subtypes in immune responses. All subtypes of IFN I signal through a unique heterodimeric receptor, interferon alpha receptor (IFNAR), composed of 2 subunits, IFNAR1 and IFNAR2. IFNR activation regulates the host response to viral infections and in adaptive immunity. Several signaling cascades can be activated by IFNR, including the Janus activated kinase-signal transducer and activation of transcription (JAK-STAT) pathway, the mitogen activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase (PI3K) pathway, the v-crk sarcoma virus CT10 oncogene homolog (avian)-like (CRKL) pathway, and NF-κB cascade. It has long been established that type I IFNs directly inhibit the proliferation of tumor cells and virus-infected cells, and increase MHC class I expression, enhancing antigen recognition. IFNs-I have also proven to be involved in immune system regulation. IFNs can either directly, through interferon receptor (IFNR), or indirectly by the induction of chemokines and cytokines, regulate the immune system. Type I IFNs enhance NK cell functions and promote survival of NK cells. Type I IFNs also affect monocytes, supporting the differentiation of monocytes into DC with high capacity for antigen presentation, and stimulate macrophage function and differentiation. Several studies also demonstrate that IFNs-I promote CD8⁺ T cell survival and functions. In some instances, it may be desirable to tune the expression of Type I IFNs using biocircuits of the present invention to avoid immunosuppression caused by long-term treatment with IFNs.

New anticancer immunotherapies are being developed that use recombinant type I IFN proteins, type I IFN transgene, type I IFN encoding vectors and type I IFN expressing cells. For example, IFN α has received approval for treatment of several neoplastic diseases, such as melanoma, RCC and multiple myeloma. Though type I IFNs are powerful tools to directly and indirectly modulate the functions of the immune system, side effects of systemic long-term treatments and lack of sufficiently high efficacy have dampened the interest of IFN α for clinical use in oncology. It is believed that if IFN levels are tightly regulated at the malignant tissues, type I IFNs are likely more efficacious. Approaches for intermittent delivery are proposed according to the observation that intermittency at an optimized pace may help to avoid signaling desensitizing mechanisms (negative feedback mechanisms) induced by IFNs-I (i.e., because of SOCS1 induction) in the responding immune cells. In accordance with the present invention, the effector module may comprise a DD-IFN fusion polypeptide. The DD and its ligand control the expression of IFN to induce an antiviral and antitumor immune responses and in the meantime, to minimize the side effects caused by long-term exposure of IFN.

In some embodiments, payloads of the present invention may comprise members of tumor necrosis factor (TNF) superfamily. The term “TNF superfamily” as used herein refers to a group of cytokines that can induce apoptosis. Members of TNF family include TNF-alpha, TNF-beta (also known as lymphotoxin-alpha (LT-α)), lymphotoxin-beta (LT-β), CD40L (CD154), CD27L (CD70), CD30L (CD153), FASL (CD178), 4-1BBL (CD137L), OX40L, TRAIL (TNF-related apoptosis inducing ligand), APRIL (a proliferation-inducing ligand), TWEAK, TRANCE, TALL-1, GITRL, LIGHT and TNFSF1 to TNFSF20 (TNF ligand superfamily member 1 to 20). In one embodiment, the payload of the invention may be TNF-alpha. TNF-alpha can cause cytolysis of tumor cells, and induce cell proliferation differentiation as well. In one aspect, the effector module of the present invention may comprise a DD-TNF alpha fusion polypeptide.

In one embodiment, the payloads of the present invention may be cytokines fused to TNF alpha ectodomain. Such payloads are produced as membrane associated cytokines fused to the TNF ectodomain. In one embodiment, the cytokine may be shed from the cell surface by the action of membrane associated proteases, and/or proteases in the extracellular space e.g. MMP9. Any of the cytokines described herein may be useful in the present invention. Such cytokine-TNF scaffold constructs may be used to preserve the native sequence of the processed cytokine while preserving regulation.

In some embodiments, payloads of the present invention may comprise inhibitory molecules that block inhibitory cytokines. The inhibitors may be blocking antibodies specific to an inhibitory cytokine, and antagonists against an inhibitory cytokine, or the like.

In some aspects, payloads of the present invention may comprise an inhibitor of a secondary cytokine IL35. IL35 belongs to the interleukin-12 (IL12) cytokine family, and is a heterodimer composed of the IL27 β chain Ebi3 and the IL12 α chain p35. Secretion of bioactive IL35 has been described only in forkhead box protein 3 (Foxp3)⁺ regulatory T cells (Tregs) (resting and activated Tregs). Unlike other membranes in the family, IL35 appears to function solely in an anti-inflammatory fashion by inhibiting effector T cell proliferation and perhaps other parameters (Collison et al., Nature, 2007, 450(7169): 566-569).

In some embodiments, payloads of the present invention may comprise inhibitors that block the transforming growth factor beta (TGF-β) subtypes (TGF-β1, TGF-β2 and TGF-β3). TGF-β is secreted by many cell types, including macrophages and is often complexed with two proteins LTBP and LAP. Serum proteinases such as plasmin catalyze the release of active TGF-β from the complex from the activated macrophages. It has been shown that an increase in expression of TGF-β correlates with the malignancy of many cancers. The immunosuppressive activity of TGF-β in the tumor microenvironment contributes to oncogenesis.

In some embodiments, payloads of the present invention may comprise inhibitors of IDO enzyme. In some embodiments, payloads fused to the DDs of the invention may be an inhibitor of an immunosuppressive molecule such as TGF-beta and IDO.

In some embodiments, payloads of the present invention may comprise chemokines and chemokine receptors. Chemokines are a family of secreted small cytokines, or signaling proteins that can induce directed chemotaxis in nearby responsive cells. The chemokine may be a SCY (small cytokine) selected from the group consisting of SCYA1-28 (CCL1-28), SCYB1-16 (CXCL1-16), SCYC1-2 (XCL1-2), SCYD-1 and SCYE-1; or a C chemokine selected from XCL1 and XCL2; or a CC chemokine selected from CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL2? and CCL28; or a CXC chemokine selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17; or a CX3C chemokine CX3CL1. In some aspects, the chemokine receptor may be a receptor for the C chemokines including XCR1; or a receptor for the CC chemokines including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 and CCR10; or a receptor for the CXC chemokines including CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5; or a CX3C chemokine receptor CX3CR1.

In some embodiments, payloads of the present invention may comprise other immunomodulators that play a critical role in immunotherapy, such as GM-CSF (Granulocyte-macrophage colony stimulating factor), erythropoietin (EPO), MIP3a, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecule (ICAM), macrophage colony stimulating factor (M-CSF), Interleukin-1 receptor activating kinase (iRAK-1), lactotransferrin, and granulocyte colony stimulating factor (G-CSF).

In some embodiments, the payload of the present invention may comprise Amphiregulin. Amphiregulin (AREG) is an EGF-like growth factor which binds to the EGFR receptor and enhances CD4+ regulatory T cells (Tregs) function. AREG promotes immune suppression in the tumor environment. Thus, in some embodiment, the payloads of the present invention may comprise Amhiregulin to dampen immune response during immunotherapy.

In some embodiments, payloads of the present invention may comprise fusion proteins wherein a cytokine, chemokine and/or other soluble factor may be fused to other biological molecules such as antibodies and or ligands for a receptor. Such fusion molecules may increase the half-life of the cytokines, reduce systemic toxicity, and increase local concentration of the cytokines at the tumor site. Fusion proteins containing two or more cytokines, chemokines and or other soluble factors may be utilized to obtain synergistic therapeutic benefits. In one embodiment, payload may be a GM-CSF/IL2 fusion protein.

In some embodiments, any of the hinge and transmembrane domains described herein may be used as a scaffold for soluble cytokine presentation. The cytokine may be operably linked to the CD8 hinge and transmembrane domain by a protease cleavage site. Cleavage at the cleavage site releases the cytokine from the cell surface membrane. In some aspects, the cytokine may be in a precursor form. Generation of the active form of the cytokine from the precursor form occurs via cleavage at the cleavage site. Any of the cytokines described herein may be engineered using the any of the hinge and transmembrane domains described herein as a scaffold.

7. Immune Regulators

In some embodiments, payloads of the present invention may comprise inhibitors (antagonists) of co-inhibitory molecules (e.g., immune checkpoint), including without limitation, PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, VISTA, BTLA, TIGIT, LAIRA, CD160, 2B4 and TGFR. In some aspects, the inhibitor may be a blocking/antagonistic antibody or fragment thereof, as discussed previously, or a ligand of the co-inhibitory receptor.

In some embodiments, payloads of the present invention may comprise agonists of co-stimulatory molecules, including without limitation, CD27, CD28, CD30, CD40, OX40 (CD134), 4-1BB (CD137), CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), GITR, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 and CD83. As a non-limiting example, agonists of co-stimulatory molecule ICOS (CD278) may be an ICOS binding protein comprising an amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5 and 6 disclosed in International Patent Publication NO: WO2016120789; the contents of which are incorporated by reference herein in their entirety.

In some aspects, the agonist of a co-stimulatory molecule may be an agonistic antibody or fragment thereof, as discussed previously; or a ligand of the co-stimulatory receptor; or any binding molecules that can enhance the biological activity of its target. For example, an agonistic ligand of OX40 may be OX40L (CD252). The OX40 ligand as used herein, includes the intact OX40 ligand, soluble OX40 ligand, fusion proteins including a functionally active portion of OX40 ligand covalently linked to a second moiety, e.g., a protein domain, and variants may be which vary in amino acid sequence from naturally occurring OX4L but which retain the ability to specifically bind to the OX40 receptor or even enhance the biological activity of OX40L.

In general, an agonist of a co-stimulatory molecule substantially enhances the biological activity of its target molecule, such as T cell activation. Desirably, the biological activity is enhanced by 10, 20, 30, 50, 70, 80, 90, 95, or even 100.

In some embodiments, payloads of the present invention may comprise immunomodulators including stress proteins and heat shock proteins (HSPs) that can integrate both innate and adaptive immune responses. They may also be other chaperones and adaptors that stimulate immune responses. As a non-limiting example, the payload of the present invention may be a fusion protein comprising an NF-KB-activating domain of Flagellin fused with an ATP-binding domain truncated glucose regulated protein 170 (Grp170) (See. US patent publication NO.: US2015/0315255; the contents of which are incorporated herein by reference in their entirety). The fusion construct forms a secretable Grp170-Flagellin hybrid chaperone (Flagrp170) which can be used to stimulate anti-cancer immune responses.

In other embodiments, the payload of the present invention may comprise a STING (stimulator of interferon gene) protein, an adaptor molecule in the cytoplasm which, as a component of the host cytosolic surveillance pathway, activates the TANK binding kinase (TBK1)-IRF3 signaling axis, resulting in the induction of IFN β and other IRF-3 dependent gene products that strongly activate innate immunity, leading to the development of an adaptive immune response consisting of both antigen-specific CD4⁺ and CD8⁺ T cells as well as pathogen-specific antibodies.

Demaria et al. reported that enforced activation of a STING protein by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), an agonist of STING, can enhance antitumor CD8⁺ T cell responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The STING-dependent antitumor immunity was dependent on type I IFNs produced by endothelial cells in the tumor microenvironment (Demaria et al., Proc. Natl. Acad. Sci. USA, 2015, 112(5): 15408-15413). These studies demonstrate that STING contributes to anti-tumor immune responses via enhancement of type I IFN signaling in the tumor microenvironment. Biocircuits, effector modules comprising STING may be applied to the tumor microenvironment to enhance anti-tumor responses either alone or in combination with other immunotherapeutic agents of the invention.

In addition to STING proteins, payloads of the present invention may comprise PRRs (pattern recognition receptors) that are involved in sensing the infection of cells by viruses and microorganisms to activate innate immune inflammatory responses. Such PRRs include Toll like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs). The RLR family is a RNA sensing system that is comprised of retinoic acid inducible gene-like-I (RIG-1), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). RIG-1 recognizes relatively short dsRNA (up to 1 kb) whereas MDA5 detects long dsRNA (more than 2 kb) to activate synthesis of type I IFNs, including IFN α and IFN β (Wilkins et al., Curr Opin Immunol., 2010, 22: 41-47). RLRs activate downstream signaling proteins evoking type I IFN production. TLRs recognize distinct structures in microbes; often referred to as “PAMPs” (pathogen associated molecular patterns). Ligand binding to TLRs invokes a cascade of intra-cellular signaling pathways that induce the production of factors involved in inflammation and immunity such as pro-inflammatory cytokines, and chemokines, as well as CD4⁺ and CD8⁺ T cell activation. Among ten TLRs identified in human, TLRs-1, 2, 4, 5 and 6 are expressed in the cell surface, while TLR-3, -7/8, and -9 are expressed with the ER compartment. In some embodiments, payloads of the invention may be one of the PRRs, or agonists of PRRs.

8. Metabolic Factors/Metabolic Checkpoint

In some embodiments, immune cells such as T cells used for immunotherapy may be metabolically reprogrammed to enhance anti-tumor T cell responses. Metabolic activities are necessary to support immune cells, specifically T cells growth, expansion, differentiation and effector functions, upon activation through T cell receptor or CAR and co-stimulatory signals. Metabolic competition between cancer cells and infiltrating immune effector cells leads to T cell energy and dysfunction.

In some embodiments, payloads of the present invention may be modulators of glycolysis. The Warburg effect in cancer cells leads to the massive generation of lactic acid that can suppress T cell cytotoxic and effector functions. As a non-limiting example, immune cells for adoptive transfer may be modified to overexpress phosphoenolpyruvate carboxykinase 1 (PCK1), which increases PEP production in T cells. Increased production of the glycolytic metabolite phosphoenolpyruvate (PEP) can repress sarco/ER Ca (2+)-ATPase (SERCA) activity, therefore sustaining T cell receptor-mediated Ca (2+)-NFAT signaling and effector functions (Ho et al, Cell, 2015, 162(6): 1217-1228).

In some embodiments, the payloads of the present invention may comprise of proteins involved in the OXPHOS pathway. For example, LEM (lymphocyte expansion molecule), a protein that can promote cytotoxic CD8⁺ T cell proliferation and effector function, and memory T cell generation in response to infection with lymphocyte choriomeningitis (CMV). LEM is part of a complex of CRIF1 (CR6 interacting factor 1) that mediates the translation and insertion of OXPHOS (Oxidative phosphorylation) proteins into mitochondrial inner membrane, thereby regulates OXPHOS activity. Thus, LEM is a positive modulator of T cell metabolism (mitochondria respiratory levels) and expansion (Okoye et al., Science, 2015, 348(6238): 995-1001).

In some embodiments, the payloads of the present invention may be inhibitors of metabolic enzymes involved in amino acid regulation. Metabolic competition for between immune cells and tumor cells can lead to establishing and maintaining an immunosuppressive tumor microenvironment due to T cell energy. Non-limiting examples include inhibitors of nitric oxide synthase and arginase I that can degrade extracellular arginine, or Indoleamine 2,3-dioxygense (IDO) that degrades tryptophan. Abrogation of these enzymes secreted by tumor cells and immune suppressive cells can promote antitumor immunity.

In other embodiments, payloads of the present invention may comprise proteins critical for de novo fatty acid and cholesterol biosynthesis. This may include proteins such as SREBP1 (also known as SREBF1), SREBP2 (SREBF2), HMGCR, HMGCS, FASN, ACACA and SQLE, and transport pathways, such as LDLR. Modulating cholesterol metabolism of cytotoxic CD8⁺ T cells may potentiate their anti-tumor effector function and proliferation. Cholesterol is a key component of membrane lipids, and has been shown that the increase in the plasma membrane cholesterol level of CD8⁺ T cells, enhances T-cell receptor clustering and signaling as well as more efficient formation of the immunological synapse (Molnar et al., J Biol Chem. 2012, 287:42664-42674). T cells used for adoptive transfer (e.g., anti-tumor CAR T cells) may be further engineered to express a protein that enhances cholesterol biosynthesis and/or transportation.

9. Safety Switch

In some embodiments, payloads of the present invention may comprise SRE regulated safety switches that can eliminate adoptively transferred cells in the case of severe toxicity, thereby mitigating the adverse effects of T cell therapy. Adoptively transferred T cells in immunotherapy may attack normal cells in response to normal tissue expression of TAA. Even on-tumor target activity of adoptively transferred T cells can result in toxicities such as tumor lysis syndrome, cytokine release syndrome and the related macrophage activation syndrome.

In one embodiment, the payloads of the present invention may eliminate the inappropriately activated cells by induction of apoptosis or by immunosurveillance have been developed in the art.

In some embodiments, payloads of the present invention may comprise inducible killer/suicide genes that acts as a safety switch. The killer/suicide gene when introduced into adoptively transferred immune cells, could control their alloreactivity. The killer/suicide gene may be an apoptotic gene (e.g., a caspase) which allows conditional apoptosis of the transduced cells by administration of a non-therapeutic ligand of the SRE (e.g., DD).

In some embodiments, the payload of the present invention may include Caspase 9. In some instances, Caspase 9 may be modified to have low basal expression and lacking the caspase recruitment domain (CARD) (SEQ ID NO.: 26 and SEQ ID NO.: 28 of US Patent No. U.S. Pat. No. 9,434,935B2; the contents of which are incorporated by reference in their entirety).

In one embodiment, the payload of the present invention is a suicide gene system, iCasp9/Chemical induced dimerization (CID) system which consists of a polypeptide derived from the Caspase9 gene fused to a drug binding domain derived from the human FK506 protein. Administration of bioinert, small molecule AP1903 (rimiducid), induces cross linking of the drug binding domains and dimerization of the fusion protein and in turn the dimerization of Caspase 9. This results in the activation of downstream effector Caspase 3 and subsequent induction of cellular apoptosis (Straathof et al., Blood, 2005, 105: 4247-4254; incorporated herein by reference in its entirety). Preclinical trials using CART including an iCasp9 gene have shown effective elimination of CAR T cells invivo in mouse models and demonstrate the potential efficacy of this approach. (Budde et al, Plos One, 2013, 8: e82742.10.1371; Hoyos et al., Leukemia, 2010; 24(6):1160-1170). In one embodiment, the payload of the invention may comprise Caspase9. In one aspect, the effector module of the invention may be a DD-Caspase9 fusion polypeptide. In some embodiments, the payload of the invention may be full length Caspase 9 (SEQ ID NO. 8364, encoded by SEQ ID No. 8365, 8366) or caspase 9 delta CD (SEQ ID NO. 8367, encoded by SEQ ID No. 8368). The Caspases 9 sequences described herein may optionally include a stop codon at the C terminal of the sequence.

In some instances, the iCasp9/CID system has been shown to have a basal rate of dimerization even in the absence of rimiducid, resulting in unintended cell death. Regulating the expression levels of iCasp9/CID is critical for maximizing the efficacy of iCasp9/CID system. Biocircuits of the present invention and/or any of their components may be utilized in regulating or tuning the iCasp9/CID system in order to optimize its utility. Other examples of proteins used in dimerization-induced apoptosis paradigm may include, but are not limited to Fas receptor, the death effector domain of Fas-associated protein, FADD, Caspase 1, Caspase 3, Caspase 7 and Caspase 8. (Belshaw P. J. et al, Chem Biol., 996, 3:731-738; MacCorkle R. A. et al, Proc Natl Acad Sci, 1998, 95:3655-3660; Spencer, D. M. et al., Curr Biol. 1996; 6:839-847; the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, the safety switch of the present invention may comprise a metabolic enzyme, such as herpes simplex virus thymidine kinase (HSV-TK) and cytosine deaminase (CD). HSV-TK phosphorylates nucleoside analogs, including acyclovir and ganciclovir (GCV) to generate triphosphate form of nucleosides. When incorporated into DNA, it leads to chain termination and cell death. Unlike the mammalian thymidine kinase, HSV-TK is characterized by 1000-fold higher affinity to nucleoside analogs such as GCV, making it suitable for use as a suicide gene in mammalian cells. Cytosine deaminase (CD) can converts 5-fluorocytosine (5-FC) into the cytotoxic 5-fluorouracil (5-FU) (Tiraby et al., FEMS Lett., 1998, 167: 41-49).

In some embodiments, the safety switch of the present invention may comprise a CYP4B1 mutant (as suicide gene), which may be co-expressed in a CAR engineered T cells (Roellecker et al., Gen Ther., 2016, May 19, doi: 10.1038/gt.2016.38).

In some embodiments, the payload of the present invention may comprise a fusion construct that can induce cell death, for example, a polypeptide with the formula of St-R1-S1-Q-S2-R2, wherein the St is a stalk sequence, R1/2 and Q are different epitopes; and S1/2 are optional spacer sequences (See, International patent publication NO.: WO2013/153391; the content of which are incorporated herein by reference in their entirety).

In some embodiments, safety switch may be mediated by therapeutic antibodies which specifically bind to an antigen that is expressed in the plasma membrane of adoptively transferred cells. The antigen-antibody interaction allows cell removal after administration of a specific monoclonal antibody against the antigen. As non-limiting examples, payloads of the present invention may comprise the antigen and antibody pair used to mediate safety switch such as CD20 and anti-CD20 antibody (Griffioen et al., Haematologica, 2009, 94:1316-1320), a protein tag and anti-tag antibody (Kieback et al., Natl. Acad. Sci. U.S.A., 2008, 105: 623-628), a compact suicide gene (RQR8) combining epitopes from CD34 (as a marker moiety) and CD20 (as a suicide moiety) which enables CD34 selection, cell tracking, as well as cell deletion after anti-CD20 monoclonal antibody administration (Philip et al., Blood, 2014, 124: 1277-1287); truncated human EGFR polypeptide and anti-EGFR monoclonal antibody (Wang et al., Blood, 2011, 118:1255-1263); and a compact polypeptide safety switch having a structural formula as discussed in U.S Patent Application Publication NO: 2015/0093401; the contents of each of which are incorporated herein by reference in their entirety.

10. Regulatory Switch

The utility of adoptive cell therapy (ACT) has been limited by the high incidence of graft versus host disease (GVHD). GVHD occurs when adoptively transferred T cells elicit an immune response resulting in host tissue damage. Recognition of host antigens by the graft cells triggers a proinflammatory cytokine storm cascade that signifies acute GVHD. GVHD is characterized as an imbalance between the effector and the regulatory arms of the immune system. In some embodiments, the payloads of the present invention may be used as regulatory switches. As used herein “regulatory switch” refers proteins, which when expressed in target cells increase tolerance to the graft by enhancing the regulatory arm of the immune system.

In one embodiment, regulatory switches may include payloads that preferentially promote the expansion of regulatory T (Treg cells). Tregs are a distinct population of cells that are positively selected on high affinity ligands in the thymus and play an important role in the tolerance to self-antigens. In addition, T regs have also been shown to play a role in peripheral tolerance to foreign antigens. Since Tregs promote immune tolerance, expansion of Tregs with the compositions of the invention may be desirable to limit GVHD.

In some embodiments, the regulatory switch may include, but is not limited to T regs activation factors such NFκB, FOXO, nuclear receptor Nr4a, Retinoic acid receptor alpha, NFAT, AP-1 and SMAD. Such factors can result in the expression of Fork headbox P3 (FOXP3) in T cells resulting in the activation of the regulatory T cell program and the expansion of T cells.

In one embodiment, the regulatory switch may be FOXP3, a transcriptional regulator in T cells. A function of FOXP3 is to suppress the function of NFAT, which leads to the suppression of expression of many genes including IL2 and effector T-cell cytokines. FOXP3 acts also as a transcription activator for genes such as CD2S, Cytotoxic T-Lymphocyte Antigen Cytotoxic T-Lymphocyte Antigen 4 (CTLA4), glucocorticoid-induced TNF receptor family gene (GITR) and folate receptor 4. FOXP3 also inhibits the differentiation of IL17 producing helper T-cells (Th17) by antagonizing RORC (RAR related orphan receptor C). Isoforms of FOXP3 lacking exon2 (FOXP3 delta 2), or exon 7 (FOXP3 delta 7) may also be used as regulatory switches. In one aspect, the effector module of the invention may be a DD-FOXP3 fusion polypeptide. FOXP3 may be a full length FOXP3 (SEQ ID NO. 8369, encoded by SEQ ID NO. 8370); or FOXP3 (amino acid 2-431 of WT) (SEQ ID NO. 8371, encoded by SEQ ID NO. 8372), delta 2 FOXP3 (SEQ ID NO. 8373, encoded by SEQ ID NO. 8374); or FOXP3 delta (amino acid 2-396 of WT) (SEQ ID NO. 8375, encoded by SEQ ID NO. 8376).

11. Homing Receptors

In some embodiments, payloads of the present invention may comprise homing receptors that guide immunotherapeutic cells to different anatomical compartments, such as designated tumor sites. For example, T cells expressing a chimeric antigen receptor may be further modified to express a homing receptor that is not normally expressed by the T cell. As used herein, the term “homing receptor” is a receptor that guides a cell expressing the receptor to a designated organ, a particular tissue, or a particular type of cell. Such trafficking receptors favor T cell accumulation in certain target organs. In some embodiments, the homing receptors of the present invention may be adhesion molecules. In other embodiments, the homing receptors of the invention may be chemokine receptors which mediate chemotaxis to chemokines. As non-limiting examples, a homing receptor may be a B cell zone homing receptor such as CXCR5; T cell zone homing receptor such as CXCR7; a gastrointestinal homing receptor such as CCR9 and integrin α4β7 (also known as lymphocyte Peyer patch adhesion molecule); a skin homing receptor such as CLA (cutaneous lymphocyte-associated antigen receptor), CCR4, CCR8 and CCR10 (See, e.g., International Patent Publication NO.: WO2016025454; the contents of which are incorporated herein by reference in their entirety). Other homing receptors include, without limitation, CXCR2 and CXCR1 which redirect chemokine receptor modified tumor-infiltrating lymphocytes to melanoma tumor (Idorn et al., Methods Mol. Biol., 2016, 1428: 261-276; and Sapoznik et al., Cancer Immunol Immunother., 2012, 61(10): 1833-1847); CCR2 which, when expressed by CD8⁺ T cells, can home modified CD8⁺ T cells to the site of prostate cancer in which the CCL2 (a CCR2 ligand) expression is increased (Garetto et al., Oncotarget, 2016, May 10. doi: 10.18632/oncotarget.9280); and CD103 as an intestinal homing receptor.

12. Immune Signaling

Treatment with immunotherapeutic agents may induce immune cell signaling, leading to the activation of cell-type specific immune activities, ultimately resulting in an immune response. In some embodiments, payloads of the present invention may be immune signaling biomolecules used to achieve exogenous control of signaling pathways. Exemplary immune signaling biomolecules include transcription factors such as Nuclear factor of activated T-cells (NFAT) (e.g., NFAT, NFAT2, NFAT3 and NFAT 4), Nuclear Factor Kappa B (NFκB), Signal transducer and activator of transcription (STAT), Activator protein-1 (AP-1), Rel, Fos, and Jun; kinases such as Janus Kinase (JAK), Extracellullar signal-regulated kinases (ERK), Mitogen-Activated Protein Kinases (MAPK), Mammalian target of rapamycin (mTOR), Phsophoinositide-dependent kinase (PDK), Protein kinase B (PKB), IkB kinase (IKK), Calcium/Calmodulin dependent kinase (CaMK); and other signaling molecules such as Ras, Cb1, Calmodulin (CaM), Calpain, and IkkB kinase.

In one embodiment, the payloads of the present invention may be administered in conjunction with inhibitors of SHP-1 and/or SHP-2. The tyrosine-protein phosphatase SHP1 (also known as PTPN6) and SHP2 (also known as PTPN11) are involved in the Programmed Cell Death (PD1) inhibitory signaling pathway. The intracellular domain of PD1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). ITSM has been shown to recruit SHP-1 and 2. This generates negative costimulatory micro clusters that induce the dephosphorylation of the proximal TCR signaling molecules, thereby resulting in suppression of T cell activation, which can lead to T cell exhaustion. In one embodiment, inhibitors of SHP-1 and 2 may include expressing dominant negative versions of the proteins in T cells, TILs or other cell types to relieve exhaustion. Such mutants can bind to the endogenous, catalytically active proteins, and inhibit their function. In one embodiment, the dominant negative mutant of SHP-1 and/or SHP-2 lack the phosphatase domain required for catalytic activity. In some embodiments, any of the dominant negative SHP-1 mutants taught Bergeron S et al. (2011). Endocrinology. 2011 December; 152(12):4581-8; Dustin J B et al. (1999) J Immunol. March 1; 162(5):2717-24; Berchtold S (1998) Mol Endocrinol. April; 12(4):556-67 and Schram et al. (2012) Am J Physiol Heart Circ Physiol. 1; 302 (1):H231-43; may be useful in the invention (the contents of each of which are incorporated by reference in their entirety).

13. Oncolytic Viruses

In some embodiments, payloads of the present invention may comprise oncolytic viruses or any components of oncolytic viruses. In some embodiments, the payload may be oncolytic viruses or components that have been genetically modified oncolytic viruses for use in oncolytic virotherapy. As used herein, the term “virotherapy” refers to a therapeutic use of oncolytic viruses (replication competent viruses) to attack and destroy cancer cells. Oncolytic viruses refer to those viruses that are able to eliminate malignancies by direct targeting and killing of cancer cells within the tumor, without causing harm to normal tissues. Exemplary oncolytic viruses and genetically engineered oncolytic viruses with cancer specific tropism may include Arvoviruses, Adenoviruses, Coxsackie viruses, Herpes Simplex Viruses (HSVs), Measles, Mumps viruses, Moloney leukemia viruses, Myxovirus, Newcastle Disease Viruses, Reoviruses, Rhabdovirus, Vesticular Stomatic Viruses, and Vaccinia Viruses (VV). It may also be chimeric viruses with increased oncolytic potential such as an adeno-parvovirus chimera in U.S. Pat. No. 9,441,246. The oncolytic viruses may be modified to be less susceptible to immune suppression while more specifically targeting particular classes of cancer cells, or be modified to insert and express cancer-suppressing transgenes. Modifications to the oncolytic virus may also be made to improve replicative potential of the virus, increase viral titers, and/or enhance the range cancer cells that can be infected by the virus. Examples of modified oncolytic viruses that may be used as payload include US Patent NO.: U.S. Pat. No. 8,282,917B2, International Patent Publication NO.: WO2011070440, WO2004078206A1, WO2016144564, WO2016119052, WO2009111892; the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, payloads of the present invention may be one or more coat proteins of the viruses, inserted transgenes, other factors that can increase intratumoral virus replication and the combinations.

In some instance, two or more oncolytic viruses may also be used as payload within the same SRE or in two or more SREs to achieve synergistic killing of target cancer cells as described in International Patent Publication NO.: WO2010020056 (the contents of which are incorporated herein by reference in their entirety).

14. Genomic Editing Systems

In some embodiments, payloads of the present invention may be components of gene editing systems including a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), CRISPR enzyme (Cas9), CRISPR-Cas9 or CRISPR system and CRISPR-CAS9 complex. It may also be other genomic editing systems, such as Zinc finger nucleases, TALEN (Transcription activator-like effector-based nucleases) and meganucleases.

Additional Features

The effector module of the present invention may further comprise a signal sequence which regulates the distribution of the payload, a cleavage and/or processing feature which facilitate cleavage of the payload from the effector module construct, a targeting and/or penetrating signal which can regulate the cellular localization of the effector module, and/or one or more linker sequences which link different components (e.g. a DD and a payload) of the effector module. In some embodiments, the effector module may further comprise of one or more additional features such as linker sequences (with specific sequences and lengths), cleavage sites, regulatory elements (that regulate expression of the protein of interest such as microRNA targeting sites), signal sequences that lead the effector module to a specific cellular or subcellular location, penetrating sequences, or tags and biomarkers for tracking the effector module.

1. Signal Sequences

In addition to the SRE (e.g., DD) and payload region, effector modules of the invention may further comprise one or more signal sequences. Signal sequences (sometimes referred to as signal peptides, targeting signals, target peptides, localization sequences, transit peptides, leader sequences or leader peptides) direct proteins (e.g., the effector module of the present invention) to their designated cellular and/or extracellular locations. Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins.

A signal sequence is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards a particular location. Signal sequences can be recognized by signal recognition particles (SRPs) and cleaved using type I and type II signal peptide peptidases. Signal sequences derived from human proteins can be incorporated as a regulatory module of the effector module to direct the effector module to a particular cellular and/or extracellular location. These signal sequences are experimentally verified and can be cleaved (Zhang et al., Protein Sci. 2004, 13:2819-2824).

In some embodiments, a signal sequence may be, although not necessarily, located at the N-terminus or C-terminus of the effector module, and may be, although not necessarily, cleaved off the desired effector module to yield a “mature” payload, i.e., an immunotherapeutic agent as discussed herein.

In some examples, a signal sequence may be a secreted signal sequence derived from a naturally secreted protein, and its variant thereof. In some instances, the secreted signal sequences may be cytokine signal sequences such as, but not limited to, IL2 signal sequence comprising amino acid of SEQ ID NO. 261 (encoded by the nucleotide sequence of SEQ ID NO. 262-265), and/or p40 signal sequence comprising the amino acid sequence of SEQ ID NO. 266 (encoded by the nucleotide sequence of SEQ ID NO. 267-275).

In some instances, signal sequences directing the payload to the surface membrane of the target cell may be used. Expression of the payload on the surface of the target cell may be useful to limit the diffusion of the payload to non-target in vivo environments, thereby potentially improving the safety profile of the payloads. Additionally, the membrane presentation of the payload may allow for physiologically and qualitative signaling as well as stabilization and recycling of the payload for a longer half-life. Membrane sequences may be the endogenous signal sequence of the N terminal component of the payload. Optionally, it may be desirable to exchange this sequence for a different signal sequence. Signal sequences may be selected based on their compatibility with the secretory pathway of the cell type of interest so that the payload is presented on the surface of the T cell. In some embodiments, the signal sequence may be IgE signal sequence comprising amino acid of SEQ ID NO. 276 (encoded by the nucleotide sequence of SEQ ID NO. 277) or CD8a signal sequence comprising amino acid SEQ ID NO. 278 (encoded by the nucleotide sequence of SEQ ID NO. 279-283).

Other examples of signal sequences include, a variant may be a modified signal sequence discussed in U.S. Pat. Nos. 8,148,494, 8,258,102, 9,133,265, 9,279,007, and U.S. patent application publication NO. 2007/0141666; and International patent application publication NO. WO1993/018181; the contents of each of which are incorporated herein by reference in their entirety. In other examples, a signal sequence may be a heterogeneous signal sequence from other organisms such as virus, yeast and bacteria, which can direct an effector module to a particular cellular site, such as a nucleus (e.g., EP 1209450). Other examples may include Aspartic Protease (NSP24) signal sequences from Trichoderma that can increase secretion of fused protein such as enzymes (e.g., U.S. Pat. No. 8,093,016 to Cervin and Kim), bacterial lipoprotein signal sequences (e.g., PCT application publication NO. 1991/09952 to Lau and Rioux), E. coli enterotoxin II signal peptides (e.g., U.S. Pat. No. 6,605,697 to Kwon et al.), E. coli secretion signal sequence (e.g., U.S. patent publication NO. 2016/090404 to Malley et al.), a lipase signal sequence from a methylotrophic yeast (e.g., U.S. Pat. No. 8,975,041), and signal peptides for DNases derived from Coryneform bacteria (e.g., U.S. Pat. No. 4,965,197); the contents of each of which are incorporated herein by reference in their entirety.

Signal sequences may also include nuclear localization signals (NLSs), nuclear export signals (NESs), polarized cell tubulo-vesicular structure localization signals (See, e.g., U.S. Pat. No. 8,993,742; Cour et al., Nucleic Acids Res. 2003, 31(1): 393-396; the contents of each of which are incorporated herein by reference in their entirety), extracellular localization signals, signals to subcellular locations (e.g. lysosome, endoplasmic reticulum, golgi, mitochondria, plasma membrane and peroxisomes, etc.) (See, e.g., U.S. Pat. No. 7,396,811; and Negi et al., Database, 2015, 1-7; the contents of each of which are incorporated herein by reference in their entirety).

2. Cleavage Sites

In some embodiments, the effector module comprises a cleavage and/or processing feature. The effector module of the present invention may include at least one protein cleavage signal/site. The protein cleavage signal/site may be located at the N-terminus, the C-terminus, at any space between the N- and the C-termini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half-way point, between the half-way point and the C-terminus, and combinations thereof.

The effector module may include one or more cleavage signal(s)/site(s) of any proteinases. The proteinases may be a serine proteinase, a cysteine proteinase, an endopeptidase, a dipeptidase, a metalloproteinase, a glutamic proteinase, a threonine proteinase and an aspartic proteinase. In some aspects, the cleavage site may be a signal sequence of furin, actinidain, calpain-1, carboxypeptidase A, carboxypeptidase P, carboxypeptidase Y, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, cathepsin B, cathepsin C, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin S, cathepsin V, clostripain, chymase, chymotrypsin, elastase, endoproteinase, enterokinase, factor Xa, formic acid, granzyme B, Matrix metallopeptidase-2, Matrix metallopeptidase-3, pepsin, proteinase K, SUMO protease, subtilisin, TEV protease, thermolysin, thrombin, trypsin and TAGZyme.

In one embodiment, the cleavage site is a furin cleavage site comprising the amino acid sequence SARNRQKRS (SEQ ID NO. 284, encoded by the nucleotide sequence of SEQ ID NO. 285), or a revised furin cleavage site comprising the amino acid sequence ARNRQKRS (SEQ ID NO. 286, encoded by the nucleotide sequence of SEQ ID NO. 287), or a modified furin site comprising the amino acid sequence ESRRVRRNKRSK (SEQ ID NO. 288, encoded by the nucleotide sequence of SEQ ID NO. 289-291).

3. Protein Tags

In some embodiments, the effector module of the invention may comprise a protein tag. The protein tag may be used for detecting and monitoring the process of the effector module. The effector module may include one or more tags such as an epitope tag (e.g., a FLAG or hemagglutinin (HA) tag). A large number of protein tags may be used for the present effector modules. They include, but are not limited to, self-labeling polypeptide tags (e.g., haloalkane dehalogenase (halotag2 or halotag7), ACP tag, clip tag, MCP tag, snap tag), epitope tags (e.g., FLAG, HA, His, and Myc), fluorescent tags (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and its variants), bioluminescent tags (e.g. luciferase and its variants), affinity tags (e.g., maltose-binding protein (MBP) tag, glutathione-S-transferase (GST) tag), immunogenic affinity tags (e.g., protein A/G, IRS, AU1, AU5, glu-glu, KT3, S-tag, HSV, VSV-G, Xpress and V5), and other tags (e.g., biotin (small molecule), StrepTag (StrepII), SBP, biotin carboxyl carrier protein (BCCP), eXact, CBP, CYD, HPC, CBD intein-chitin binding domain, Trx, NorpA, and NusA.

In other embodiments, a tag may also be selected from those disclosed in U.S. Pat. Nos. 8,999,897; 8,357,511; 7,094,568; 5,011,912; 4,851,341; and 4,703,004; U.S patent application publication NOs.: 2013/115635 and 2013/012687; and International application publication NO.: WO2013/091661; the contents of each of which are incorporated herein by reference in their entirety.

In some aspects, a multiplicity of protein tags, either the same or different tags, may be used; each of the tags may be located at the same N- or C-terminus, whereas in other cases these tags may be located at each terminus.

4. Targeting Peptides

In some embodiments, the effector module of the invention may further comprise a targeting and/or penetrating peptide. Small targeting and/or penetrating peptides that selectively recognize cell surface markers (e.g. receptors, trans-membrane proteins, and extra-cellular matrix molecules) can be employed to target the effector module to the desired organs, tissues or cells. Short peptides (5-50 amino acid residues) synthesized in vitro and naturally occurring peptides, or analogs, variants, derivatives thereof, may be incorporated into the effector module for homing the effector module to the desired organs, tissues and cells, and/or subcellular locations inside the cells.

In some embodiments, a targeting sequence and/or penetrating peptide may be included in the effector module to drive the effector module to a target organ, or a tissue, or a cell (e.g., a cancer cell). In other embodiments, a targeting and/or penetrating peptide may direct the effector module to a specific subcellular location inside a cell.

A targeting peptide has any number of amino acids from about 6 to about 30 inclusive. The peptide may have 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Generally, a targeting peptide may have 25 or fewer amino acids, for example, 20 or fewer, for example 15 or fewer.

Exemplary targeting peptides may include, but are not limited to, those disclosed in the art, e.g., U.S. Pat. Nos. 9,206,231, 9,110,059, 8,706,219; and 8,772,449, and U.S. application publication NOs. 2016/089447. 2016/060296. 2016/060314. 2016/060312. 2016/060311. 2016/009772. 2016/002613. 2015/314011 and 2015/166621. and International application publication NOs. WO2015/179691 and WO2015/183044; the contents of each of which are incorporated herein by reference in their entirety.

5. Linkers

In some embodiments, the effector module of the invention may further comprise a linker sequence. The linker region serves primarily as a spacer between two or more polypeptides within the effector module. A “linker” or “spacer”, as used herein, refers to a molecule or group of molecules that connects two molecules, or two parts of a molecule such as two domains of a recombinant protein.

In some embodiments, “Linker” (L) or “linker domain” or “linker region” or “linker module” or “peptide linker” as used herein refers to an oligo- or polypeptide region of from about 1 to 100 amino acids in length, which links together any of the domains/regions of the effector module (also called peptide linker). The peptide linker may be 1-40 amino acids in length, or 2-30 amino acids in length, or 20-80 amino acids in length, or 50-100 amino acids in length. Linker length may also be optimized depending on the type of payload utilized and based on the crystal structure of the payload. In some instances, a shorter linker length may be preferably selected. In some aspects, the peptide linker is made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Serine (S), Cysteine (C), Threonine (T), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Histidine (H), Lysine (K), Arginine (R), Aspartate (D), Glutamic acid (E), Asparagine (N), and Glutamine (Q). One or more of these amino acids may be glycosylated, as is understood by those in the art. In some aspects, amino acids of a peptide linker may be selected from Alanine (A), Glycine (G), Proline (P), Asparagine (R), Serine (S), Glutamine (Q) and Lysine (K).

In one example, an artificially designed peptide linker may preferably be composed of a polymer of flexible residues like Glycine (G) and Serine (S) so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not interfere with one another. The choice of a particular linker sequence may concern if it affects biological activity, stability, folding, targeting and/or pharmacokinetic features of the fusion construct. Examples of peptide linkers include, but are not limited to: SG, MH, GGSG (SEQ ID NO. 292, encoded by the nucleotide sequence of SEQ ID NO. 293; GGSGG (SEQ ID NO. 294), encoded by the nucleotide sequence of SEQ ID NOs. 295-299; GGSGGG (SEQ ID NO. 78), encoded by the nucleotide sequence of SEQ ID NO. 93 and 300; SGGGS (SEQ ID NO. 301), encoded by the nucleotide sequence of SEQ ID NO. 302-303; GGSGGGSGG (SEQ ID NO. 77), encoded by the nucleotide sequence of SEQ ID NO. 92; GGGGG (SEQ ID NO. 304), GGGGS (SEQ ID NO. 305) or (GGGGS)n (n=2 (SEQ ID NO. 306), 3 (SEQ ID NO. 307; encoded by SEQ ID NO. 308-313), 4 (SEQ ID NO. 314), 5 (SEQ ID NO. 315), or 6 (SEQ ID NO. 316)), SSSSG (SEQ ID NO. 317) or (SSSSG)n (n=2 (SEQ ID NO. 318), 3 (SEQ ID NO. 319), 4 (SEQ ID NO. 320), 5 (SEQ ID NO. 321), or 6 (SEQ ID NO. 322)), SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO. 323), encoded by the nucleotide sequence of SEQ ID NO. 324; EFSTEF (SEQ ID NO. 325), encoded by the nucleotide sequence of SEQ ID NOs. 326-327; GKSSGSGSESKS (SEQ ID NO. 328), GGSTSGSGKSSEGKG (SEQ ID NO. 329), GSTSGSGKSSSEGSGSTKG (SEQ ID NO. 330), GSTSGSGKPGSGEGSTKG (SEQ ID NO. 331), VDYPYDVPDYALD (SEQ ID NO. 332), encoded by the nucleotide sequence of SEQ ID NO. 333; or EGKSSGSGSESKEF (SEQ ID NO. 334); or GSGSGS (SEQ ID NO. 8330), encoded by the nucleotide sequence of SEQ ID NO. 8347; or GSGSGSGS (SEQ ID NO. 8331), encoded by the nucleotide sequence of SEQ ID NO. 8348; or GSGSGGGSGS (SEQ ID NO. 8332), encoded by the nucleotide sequence of SEQ ID NO. 8349; SGSGSGS linker (SEQ ID NO: 8382), or SG linker, encoded by AGTGGT; or an LD Linker comprising Lysine Aspartic acid, encoded by CTAGAT. Linkers may also be DNA restriction enzyme recognition sites or modifications thereof such as flexible GS or G/S rich linker; BamH1 Site encoded by GGATCC; flexible G/S rich linker or BamH1 Site; SR/Xba I site, encoded by TCTAGA; or a GSG linker (BamH1-Gly) linker, encoded by GGATCCGGA.

In other examples, a peptide linker may be made up of a majority of amino acids that are sterically unhindered, such as Glycine (G) and Alanine (A). Exemplary linkers are polyglycines (such as (G)₄ (SEQ ID NO: 8378), (G)₅ (SEQ ID NO: 8379), (G)₈ (SEQ ID NO: 8380)), poly(GA), and polyalanines. The linkers described herein are exemplary, and linkers that are much longer and which include other residues are contemplated by the present invention.

A linker sequence may be a natural linker derived from a multi-domain protein. A natural linker is a short peptide sequence that separates two different domains or motifs within a protein.

In some aspects, linkers may be flexible or rigid. In other aspects, linkers may be cleavable or non-cleavable. As used herein, the terms “cleavable linker domain or region” or “cleavable peptide linker” are used interchangeably. In some embodiments, the linker sequence may be cleaved enzymatically and/or chemically. Examples of enzymes (e.g., proteinase/peptidase) useful for cleaving the peptide linker include, but are not limited, to Arg-C proteinase, Asp-N endopeptidase, chymotrypsin, clostripain, enterokinase, Factor Xa, glutamyl endopeptidase, Granzyme B, Achromobacter proteinase I, pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, trypsin, and members of the Caspase family of proteolytic enzymes (e.g. Caspases 1-10). Chemical sensitive cleavage sites may also be included in a linker sequence. Examples of chemical cleavage reagents include, but are not limited to, cyanogen bromide, which cleaves methionine residues; N-chloro succinimide, iodobenzoic acid or BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], which cleaves tryptophan residues; dilute acids, which cleave at aspartyl-prolyl bonds; and e aspartic acid-proline acid cleavable recognition sites (i.e., a cleavable peptide linker comprising one or more D-P dipeptide moieties). The fusion module may include multiple regions encoding peptides of interest separated by one or more cleavable peptide linkers.

In other embodiments, a cleavable linker may be a “self-cleaving” linker peptide, such as 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof. In some embodiments, the biocircuits of the present invention may include 2A peptides. The 2A peptide is a sequence of about 20 amino acid residues from a virus that is recognized by a protease (2A peptidases) endogenous to the cell. The 2A peptide was identified among picornaviruses, a typical example of which is the Foot- and Mouth disease virus (Robertson B H, et. al., J Virol 1985, 54:651-660). 2A-like sequences have also been found in Picornaviridae like equine rhinitis A virus, as well as unrelated viruses such as porcine teschovirus-1 and the insect Thosea asigna virus (TaV). In such viruses, multiple proteins are derived from a large polyprotein encoded by an open reading frame. The 2A peptide mediates the co-translational cleavage of this polyprotein at a single site that forms the junction between the virus capsid and replication polyprotein domains. The 2A sequences contain the consensus motif D-V/I-E-X-N-P-G-P (SEQ ID NO: 8381). These sequences are thought to act co-translationally, preventing the formation of a normal peptide bond between the glycine and last proline, resulting in the ribosome skipping of the next codon (Donnelly M L et al. (2001). J Gen Virol, 82:1013-1025). After cleavage, the short peptide remains fused to the C-terminus of the protein upstream of the cleavage site, while the proline is added to the N-terminus of the protein downstream of the cleavage site. Of the 2A peptides identified, four have been widely used namely FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); porcine teschovirus-1 2A (P2A) and Thoseaasigna virus 2A (T2A). In some embodiments, the 2A peptide sequences useful in the present invention are selected from SEQ ID NO. 8-11 of International Patent Publication WO2010042490, the contents of which are incorporated by reference in its entirety. In some embodiments, the cleavage site may be a P2A cleavable peptide (SEQ ID NO. 8239), encoded by the nucleotide sequence of SEQ ID NO. 8269.

Other linkers will be apparent to those skilled in the art and may be used in connection with alternate embodiments of the invention.

The linkers of the present invention may also be non-peptide linkers. For example, alkyl linkers such as —NH—(CH₂) a-C(O)—, wherein a=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc.

In some aspects, the linker may be an artificial linker from U.S. Pat. Nos. 4,946,778, 5,525,491, 5,856,456, and International patent publication NO. WO2012/083424; the contents of each of which are incorporated herein by reference in their entirety.

6. Embedded Stimulus, Signals and Other Regulatory Features

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bind to the 3′UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. The polynucleotides of the invention may comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNA such as those taught in US Publication No. US2005/0261218 and US Publication No. US2005/0059005, the contents of which are incorporated herein by reference in their entirety. As a non-limiting embodiment, known microRNAs, their sequences and their binding site sequences in the human genome are listed Table 14 of the co-owned U.S. Provisional Patent Application No. 62/320,864 filed Apr. 11, 2016, 62/466,596 filed Mar. 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety).

A microRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence. A microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA. In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1. In some embodiments, a microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1. See for example, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P, Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of the microRNA seed have complete complementarity with the target sequence. By engineering microRNA target sequences into the polynucleotides encoding the biocircuit components, effector modules, SREs or payloads of the invention one can target the molecule for degradation or reduced translation, provided the microRNA in question is available. This process will reduce the hazard of off target effects upon nucleic acid molecule delivery.

Identification of microRNA, microRNA target regions, and their expression patterns and role in biology have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner and Naldini, Tissue Antigens. 2012 80:393-403 and all references therein; each of which is herein incorporated by reference in its entirety).

For example, if the polynucleotide is not intended to be delivered to the liver but ends up there, then miR-122, a microRNA abundant in liver, can inhibit the expression of the polynucleotide if one or multiple target sites of miR-122 are engineered into the polynucleotide. Introduction of one or multiple binding sites for different microRNA can be engineered to further decrease the longevity, stability, and protein translation of a polynucleotide hence providing an additional layer of tenability beyond the stimulus selection, SRE design and payload variation.

As used herein, the term “microRNA site” refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that “binding” may follow traditional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.

Conversely, for the purposes of the polynucleotides of the present invention, microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, miR-122 binding sites may be removed to improve protein expression in the liver.

Regulation of expression in multiple tissues can be accomplished through introduction or removal or one or several microRNA binding sites.

Specifically, microRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APCs) (e.g. dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc. Immune cell specific microRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cells specific microRNAs also regulate many aspects of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells). For example, miR-142 and miR-146 are exclusively expressed in the immune cells, particularly abundant in myeloid dendritic cells. Introducing the miR-142 binding site into the 3′-UTR of a polypeptide of the present invention can selectively suppress the gene expression in the antigen presenting cells through miR-142 mediated mRNA degradation, limiting antigen presentation in professional APCs (e.g. dendritic cells) and thereby preventing antigen-mediated immune response after gene delivery (see, Annoni A et al., blood, 2009, 114, 5152-5161, the content of which is herein incorporated by reference in its entirety.)

In one embodiment, microRNAs binding sites that are known to be expressed in immune cells, in particular, the antigen presenting cells, can be engineered into the polynucleotides to suppress the expression of the polynucleotide in APCs through microRNA mediated RNA degradation, subduing the antigen-mediated immune response, while the expression of the polynucleotide is maintained in non-immune cells where the immune cell specific microRNAs are not expressed.

Many microRNA expression studies have been conducted, and are described in the art, to profile the differential expression of microRNAs in various cancer cells/tissues and other diseases. Some microRNAs are abnormally over-expressed in certain cancer cells and others are under-expressed. For example, microRNAs are differentially expressed in cancer cells (WO2008/154098, US2013/0059015, US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224); pancreatic cancers and diseases (US2009/0131348, US2011/0171646, US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation (U.S. Pat. No. 8,415,096); prostate cancer (US2013/0053264); hepatocellular carcinoma (WO2012/151212, US2012/0329672, WO2008/054828, U.S. Pat. No. 8,252,538); lung cancer cells (WO2011/076143, WO2013/033640, WO2009/070653, US2010/0323357); cutaneous T cell lymphoma (WO2013/011378); colorectal cancer cells (WO2011/0281756, WO2011/076142); cancer positive lymph nodes (WO2009/100430, US2009/0263803); nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary disease (US2012/0264626, US2013/0053263); thyroid cancer (WO2013/066678); ovarian cancer cells (US2012/0309645, WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740, US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974, US2012/0316081, US2012/0283310, WO2010/018563, the content of each of which is incorporated herein by reference in their entirety).

In one embodiment, microRNA may be used as described herein in support of the creation of tunable biocircuits.

In some embodiments, effector modules may be designed to encode (as a DNA or RNA or mRNA) one or more payloads, SREs and/or regulatory sequence such as a microRNA or microRNA binding site. In some embodiments, any of the encoded payloads or SREs may be stabilized or de-stabilized by mutation and then combined with one or more regulatory sequences to generate a dual or multi-tuned effector module or biocircuit system.

Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature. For example, an SRE may represent a destabilizing domain, while mutations in the protein payload may alter its cleavage sites or dimerization properties or half-life and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features. Consequently, the present invention embraces biocircuits which are multifactorial in their tenability.

In some embodiments, compositions of the invention may include optional proteasome adaptors. As used herein, the term “proteasome adaptor” refers to any nucleotide/amino acid sequence that targets the appended payload for degradation. In some aspects, the adaptors target the payload for degradation directly thereby circumventing the need for ubiquitination reactions. Proteasome adaptors may be used in conjunction with destabilizing domains to reduce the basal expression of the payload. Exemplary proteasome adaptors include the UbL domain of Rad23 or hHR23b, HPV E7 which binds to both the target protein Rb and the S4 subunit of the proteasome with high affinity, which allows direct proteasome targeting, bypassing the ubiquitination machinery; the protein gankyrin which binds to Rb and the proteasome subunit S6.

Such biocircuits may be engineered to contain one, two, three, four or more tuned features.

Polynucleotides

The present invention provides polynucleotides encoding novel hPDE5 DDs, effector modules comprising payloads and associated DDs, biocircuit systems comprising DDs and effector modules, and other components of the present invention.

The invention provides isolated biocircuit polypeptides, effector modules, stimulus response elements (SREs) and payloads, as well as polynucleotides encoding any of the foregoing; vectors comprising polynucleotides of the invention; and cells expressing polypeptides, polynucleotides and vectors of the invention. The polypeptides, polynucleotides, viral vectors and cells are useful for inducing anti-tumor immune responses in a subject.

The term “polynucleotide” or “nucleic acid molecule” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides, e.g., linked nucleosides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.

In some embodiments, polynucleotides of the invention may be a messenger RNA (mRNA) or any nucleic acid molecule and may or may not be chemically modified. In one aspect, the nucleic acid molecule is a mRNA. As used herein, the term “messenger RNA (mRNA)” refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Building on this wild type modular structure, the present invention expands the scope of functionality of traditional mRNA molecules by providing payload constructs which maintain a modular organization, but which comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide, for example tenability of function. As used herein, a “structural” feature or modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleosides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.

In some embodiments, polynucleotides of the present invention may harbor 5′UTR sequences which play a role in translation initiation. 5′UTR sequences may include features such as Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of genes, Kozak sequences have the consensus XCCR (A/G) CCAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) and X is any nucleotide. In one embodiment, the Kozak sequence is ACCGCC. By engineering the features that are typically found in abundantly expressed genes of target cells or tissues, the stability and protein production of the polynucleotides of the invention can be enhanced.

Further provided are polynucleotides, which may contain an internal ribosome entry site (IRES) which play an important role in initiating protein synthesis in the absence of 5′ cap structure in the polynucleotide. An IRES may act as the sole ribosome binding site, or may serve as one of the multiple binding sites. Polynucleotides of the invention containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes giving rise to bicistronic and/or multicistronic nucleic acid molecules.

In some embodiments, polynucleotides encoding biocircuits, effector modules, DDs and payloads may include from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides). In some aspects, polynucleotides of the invention may include more than 10,000 nucleotides.

Regions of the polynucleotides which encode certain features such as cleavage sites, linkers, trafficking signals, tags or other features may range independently from 10-1,000 nucleotides in length (e.g., greater than 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

In some embodiments, polynucleotides of the present invention may further comprise embedded regulatory moieties such as microRNA binding sites within the 3′UTR of nucleic acid molecules which when bind to microRNA molecules, down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. Conversely, for the purposes of the polynucleotides of the present invention, microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, miR-142 and miR-146 binding sites may be removed to improve protein expression in the immune cells. In some embodiments, any of the encoded payloads may be may be regulated by an SRE and then combined with one or more regulatory sequences to generate a dual or multi-tuned effector module or biocircuit system.

In some embodiments, polynucleotides of the present invention may encode fragments, variants, derivatives of polypeptides of the inventions. In some aspects, the variant sequence may keep the same or a similar activity. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to the start sequence. Generally, variants of a particular polynucleotide or polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res., 1997, 25:3389-3402.)

In some embodiments, polynucleotides of the present invention may be modified. As used herein, the terms “modified”, or as appropriate, “modification” refers to chemical modification with respect to A, G, U (T in DNA) or C nucleotides. Modifications may be on the nucleoside base and/or sugar portion of the nucleosides which comprise the polynucleotide. In some embodiments, multiple modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase and the sugar. Modifications to the polynucleotides of the present invention may include any of those taught in, for example, International Publication NO. WO2013/052523, the contents of which are incorporated herein by reference in its entirety.

As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group.

In some embodiments, the modification may be on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). Other modifications which may be used are taught in, for example, International Application NO.: WO2013/052523, the contents of which are incorporated herein by reference in their entirety.

Chemical modifications and/or substitution of the nucleotides or nucleobases of the polynucleotides of the invention which are useful in the present invention include any modified substitutes known in the art, for example, (±)1-(2-Hydroxypropyl)pseudouridine TP, (2R)-1-(2-Hydroxypropyl)pseudouridine TP, 1-(4-Methoxy-phenyl)pseudo-UTP, 2′-O-dimethyladenosine, 1,2′-O-dimethylguanosine, 1,2′-O-dimethylinosine, 1-Hexyl-pseudo-UTP, 1-Homoallylpseudouridine TP, 1-Hydroxymethylpseudouridine TP, 1-iso-propyl-pseudo-UTP, 1-Me-2-thio-pseudo-UTP, 1-Me-4-thio-pseudo-UTP, 1-Me-alpha-thio-pseudo-UTP, 1-Me-GTP, 2′-Amino-2′-deoxy-ATP, 2′-Amino-2′-deoxy-CTP, 2′-Amino-2′-deoxy-GTP, 2′-Amino-2′-deoxy-UTP, 2′-Azido-2′-deoxy-ATP, tubercidine, undermodified hydroxywybutosine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, wybutosine, wyosine, xanthine, Xanthosine-5′-TP, xylo-adenosine, zebularine, α-thio-adenosine, α-thio-cytidine, α-thio-guanosine, and/or α-thio-uridine.

Polynucleotides of the present invention may comprise one or more of the modifications taught herein. Different sugar modifications, base modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the polynucleotide of the invention. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. A modification may also be a 5′ or 3′ terminal modification. The polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

In some embodiments, one or more codons of the polynucleotides of the present invention may be replaced with other codons encoding the native amino acid sequence to tune the expression of the SREs, through a process referred to as codon selection. Since mRNA codon, and tRNA anticodon pools tend to vary among organisms, cell types, sub cellular locations and over time, the codon selection described herein is a spatiotemporal (ST) codon selection.

In some embodiments of the invention, certain polynucleotide features may be codon optimized. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cell by replacing at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons of the native sequence with codons that are most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage may be measured using the Codon Adaptation Index (CAI) which measures the deviation of a coding polynucleotide sequence from a reference gene set. Codon usage tables are available at the Codon Usage Database (www.kazusa.or.jp/codon/) and the CAI can be calculated by EMBOSS CAI program (emboss.sourceforge.net/). Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias nucleotide content to alter stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein signaling sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.), OptimumGene (GenScript, Piscataway, N.J.), algorithms such as but not limited to, DNAWorks v3.2.3, and/or proprietary methods. In one embodiment, a polynucleotide sequence or portion thereof is codon optimized using optimization algorithms. Codon options for each amino acid are well-known in the art as are various species table for optimizing for expression in that particular species.

In some embodiments of the invention, certain polynucleotide features may be codon optimized. For example, a preferred region for codon optimization may be upstream (5′) or downstream (3′) to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon optimization of the payload encoding region or open reading frame (ORF).

After optimization (if desired), the polynucleotide components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.

Spatiotemporal codon selection may impact the expression of the polynucleotides of the invention, since codon composition determines the rate of translation of the mRNA species and its stability. For example, tRNA anticodons to optimized codons are abundant, and thus translation may be enhanced. In contrast, tRNA anticodons to less common codons are fewer and thus translation may proceed at a slower rate. Presnyak et al. have shown that the stability of an mRNA species is dependent on the codon content, and higher stability and thus higher protein expression may be achieved by utilizing optimized codons (Presnyak et al. (2015) Cell 160, 1111-1124; the contents of which are incorporated herein by reference in their entirety). Thus, in some embodiments, ST codon selection may include the selection of optimized codons to enhance the expression of the SRES, effector modules and biocircuits of the invention. In other embodiments, spatiotemporal codon selection may involve the selection of codons that are less commonly used in the genes of the host cell to decrease the expression of the compositions of the invention. The ratio of optimized codons to codons less commonly used in the genes of the host cell may also be varied to tune expression.

In some embodiments, certain regions of the polynucleotide may be modified using codon selection methods. For example, a preferred region for codon selection may be upstream (5′) or downstream (3′) to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon selection of the payload encoding region or open reading frame (ORF).

The stop codon of the polynucleotides of the present invention may be modified to include sequences and motifs to alter the expression levels of the SREs, payloads and effector modules of the present invention. Such sequences may be incorporated to induce stop codon readthrough, wherein the stop codon may specify amino acids e.g. selenocysteine or pyrrolysine. In other instances, stop codons may be skipped altogether to resume translation through an alternate open reading frame. Stop codon read through may be utilized to tune the expression of components of the effector modules at a specific ratio (e.g. as dictated by the stop codon context). Examples of preferred stop codon motifs include UGAN, UAAN, and UAGN, where N is either C or U.

Polynucleotide modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis and recombinant technology. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.

In some embodiments, polynucleotides of the invention may comprise two or more effector module sequences, or two or more payload sequences, which are in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different effector module component.

In yet another embodiment, polynucleotides of the invention may comprise two or more effector module component sequences with each component having one or more SRE sequences (DD sequences), or two or more payload sequences. As a non-limiting example, the sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times in each of the regions. As another non-limiting example, the sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times across the entire polynucleotide. In these patterns, each letter, A, B, or C represent a different sequence or component.

According to the present invention, polynucleotides encoding distinct biocircuits, effector modules, SREs and payload constructs may be linked together through the 3′-end using nucleotides which are modified at the 3′-terminus. Chemical conjugation may be used to control the stoichiometry of delivery into cells. Polynucleotides can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug. As non-limiting examples, they may be conjugates with other immune conjugates.

In some embodiments, the compositions of the polynucleotides of the invention may generated by combining the various components of the effector modules using the Gibson assembly method. The Gibson assembly reaction consists of three isothermal reactions, each relying on a different enzymatic activity including a 5′ exonuclease which generates long overhangs, a polymerase which fills in the gaps of the annealed single strand regions and a DNA ligase which seals the nicks of the annealed and filled-in gaps. Polymerase chain reactions are performed prior to Gibson assembly which may be used to generate PCR products with overlapping sequence. These methods can be repeated sequentially, to assemble larger and larger molecules. For example, the method can comprise repeating a method as above to join a second set of two or more DNA molecules of interest to one another, and then repeating the method again to join the first and second set DNA molecules of interest, and so on. At any stage during these multiple rounds of assembly, the assembled DNA can be amplified by transforming it into a suitable microorganism, or it can be amplified in vitro (e.g., with PCR).

In some embodiments, polynucleotides of the invention may encode effector modules comprising a destabilizing domain (DD) and at least one payload taught herein. The DD domain may be a hPDE5 mutant comprising one, two, three, four, five or more mutations

In some embodiments, the effector module may be a PDE5-GFP fusion encoded by SEQ ID NO. 95-106; 205-222; 234-236; 256-260; 378-379; 469-503; and 526-533. In some embodiments, the effector module may be hPDE5-CAR constructs, encoded by SEQ ID NO. 8285-8298 or a hPDE5-IL15-IL15Ra constructs, encoded by SEQ ID NO. 8352-8361.

Cells

In accordance with the present invention, cells genetically modified to express at least one biocircuit, SRE (e. g, DD), effector module and immunotherapeutic agent of the invention, are provided. Cells of the invention may include, without limitation, immune cells, stem cells and tumor cells. In some embodiments, immune cells are immune effector cells, including, but not limiting to, T cells such as CD8⁺ T cells and CD4⁺ T cells (e.g., Th1, Th2, Th17, Foxp3+ cells), memory T cells such as T memory stem cells, central T memory cells, and effector memory T cells, terminally differentiated effector T cells, natural killer (NK) cells, NK T cells, tumor infiltrating lymphocytes (TILs), cytotoxic T lymphocytes (CTLs), regulatory T cells (Tregs), and dendritic cells (DCs), other immune cells that can elicit an effector function, or the mixture thereof. T cells may be Tαβ cells and Tγδ cells. In some embodiments, stem cells may be from human embryonic stem cells, mesenchymal stem cells, and neural stem cells. In some embodiments, T cells may be depleted endogenous T cell receptors (See U.S. Pat. Nos. 9,273,283; 9,181,527; and 9,028,812; the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, cells of the invention may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.

In some embodiments, cells of the invention may be mammalian cells, particularly human cells. Cells of the invention may be primary cells or immortalized cell lines.

In some embodiments, cells of the invention may include expansion factors as payload to trigger proliferation and expansion of the cells. Exemplary payloads include RAS such as KRAS, NRAS, RRAS, RRAS2, MRAS, ERAS, and HRAS, DIRAS such as DIRAS1, DIRAS2, and DIRAS3, NKIRAS such as NKIRAS1, and NKIRAS2, RAL such as RALA, and RALB, RAP such as RAP1A, RAP1B, RAP2A, RAP2B, and RAP2C, RASD such as RASD1, and RASD2, RASL such as RASL10A, RASL10B, RASL11A, RASL11B, and RASL12, REM such as REM1, and REM2, GEM, RERG, RERGL, and RRAD.

Engineered immune cells can be accomplished by transducing a cell compositions with a polypeptide of a biocircuit, an effector module, a SRE and/or a payload of interest (i.e., immunotherapeutic agent), or a polynucleotide encoding said polypeptide, or a vector comprising said polynucleotide. The vector may be a viral vector such as a lentiviral vector, a gamma-retroviral vector, a recombinant AAV, an adenoviral vector and an oncolytic viral vector. In other aspects, non-viral vectors for example, nanoparticles and liposomes may also be used. In some embodiments, immune cells of the invention are genetically modified to express at least one immunotherapeutic agent of the invention which is tunable using a stimulus. In some examples, two, three or more immunotherapeutic agents constructed in the same biocircuit and effector module are introduced into a cell. In other examples, two, three, or more biocircuits, effector modules, each of which comprises an immunotherapeutic agent, may be introduced into a cell.

In some embodiments, immune cells of the invention may be T cells modified to express an antigen-specific T cell receptor (TCR), or an antigen specific chimeric antigen receptor (CAR) taught herein (known as CAR T cells). Accordingly, at least one polynucleotide encoding a CAR system (or a TCR) described herein, or a vector comprising the polynucleotide is introduced into a T cell. The T cell expressing the CAR or TCR binds to a specific antigen via the extracellular targeting moiety of the CAR or TCR, thereby a signal via the intracellular signaling domain (s) is transmitted into the T cell, and as a result, the T cell is activated. The activated CAR T cell changes its behavior including release of a cytotoxic cytokine (e.g., a tumor necrosis factor, and lymphotoxin, etc.), improvement of a cell proliferation rate, change in a cell surface molecule, or the like. Such changes cause destruction of a target cell expressing the antigen recognized by the CAR or TCR. In addition, release of a cytokine or change in a cell surface molecule stimulates other immune cells, for example, a B cell, a dendritic cell, a NK cell, and a macrophage.

The CAR introduced into a T cell may be a first-generation CAR including only the intracellular signaling domain from TCR CD3zeta, or a second-generation CAR including the intracellular signaling domain from TCR CD3zeta and a costimulatory signaling domain, or a third-generation CAR including the intracellular signaling domain from TCR CD3zeta and two or more costimulatory signaling domains, or a split CAR system, or an on/off switch CAR system. In one example, the expression of the CAR or TCR is controlled by a destabilizing domain (DD) such as a hDHFR mutant, in the effector module of the invention. The presence or absence of hDHFR binding ligand such as TMP is used to tune the CAR or TCR expression in transduced T cells or NK cells.

In some embodiments, CAR T cells of the invention may be further modified to express another one, two, three or more immunotherapeutic agents. The immunotherapeutic agents may be another CAR or TCR specific to a different target molecule; a cytokine such as IL2, IL12, IL15 and IL18, or a cytokine receptor such as IL15Rα; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; or a safety switch gene (e.g., a suicide gene) that kills activated T cells when a severe event is observed after adoptive cell transfer or when the transferred immune cells are no-longer needed. These molecules may be included in the same effector module or in separate effector modules.

In one embodiment, the CAR T cell (including TCR T cell) of the invention may be an “armed” CAR T cell which is transformed with an effector module comprising a CAR and an effector module comprising a cytokine. The inducible or constitutively secrete active cytokines further armor CAR T cells to improve efficacy and persistence. In this context, such CAR T cell is also referred to as “armored CAR T cell”. The “armor” molecule may be selected based on the tumor microenvironment and other elements of the innate and adaptive immune systems. In some embodiments, the molecule may be a stimulatory factor such as IL2, IL12, IL15, IL18, type I IFN, CD40L and 4-1BBL which have been shown to further enhance CAR T cell efficacy and persistence in the face of a hostile tumor microenvironment via different mechanisms (Yeku et al., Biochem Soc Trans., 2016, 44(2): 412-418).

In some aspects, the armed CAR T cell of the invention is modified to express a CD19 CAR and IL12. Such T cells, after CAR mediated activation in the tumor, release inducible IL12 which augments T-cell activation and attracts and activates innate immune cells to eliminate CD19-negative cancer cells.

In one embodiment, T cells of the invention may be modified to express an effector module comprising a CAR and an effector module comprising a suicide gene.

In one embodiment, the CAR T cell (including TCR T cell) of the invention may be transformed with effector modules comprising a cytokine and a safety switch gene (e.g., suicide gene). The suicide gene may be an inducible caspase such as caspase 9 which induces apoptosis, when activated by an extracellular stimulus of a biocircuit system. Such induced apoptosis eliminates transferred cell as required to decrease the risk of direct toxicity and uncontrolled cell proliferation.

In some embodiments, immune cells of the invention may be NK cells modified to express an antigen-specific T cell receptor (TCR), or an antigen specific chimeric antigen receptor (CAR) taught herein.

Natural killer (NK) cells are members of the innate lymphoid cell family and characterized in humans by expression of the phenotypic marker CD56 (neural cell adhesion molecule) in the absence of CD3 (T-cell co-receptor). NK cells are potent effector cells of the innate immune system which mediate cytotoxic attack without the requirement of prior antigen priming, forming the first line of defense against diseases including cancer malignancies and viral infection.

Several pre-clinical and clinical trials have demonstrated that adoptive transfer of NK cells is a promising treatment approach against cancers such as acute myeloid leukemia (Ruggeri et al., Science; 2002, 295: 2097-2100; and Geller et al., Immunotherapy, 2011, 3: 1445-1459). Adoptive transfer of NK cells expressing CAR such as DAP12-Based Activating CAR revealed improved eradication of tumor cells (Topfer et al., J Immunol. 2015; 194:3201-3212). NK cell engineered to express a CS-1 specific CAR also displayed enhanced cytolysis and interferon-γ (IFN γ) production in multiple myeloma (Chu et al., Leukemia, 2014, 28(4): 917-927).

NK cell activation is characterized by an array of receptors with activating and inhibitory functions. The important activation receptors on NK cells include CD94/NKG2C and NKG2D (the C-type lectin-like receptors), and the natural cytotoxicity receptors (NCR) NKp30, NKp44 and NKp46, which recognize ligands on tumor cells or virally infected cells. NK cell inhibition is essentially mediated by interactions of the polymorphic inhibitory killer cell immunoglobulin-like receptors (KIRs) with their cognate human-leukocyte-antigen (HLA) ligands via the alpha-1 helix of the HLA molecule. The balance between signals that are generated from activating receptors and inhibitory receptors mainly determines the immediate cytotoxic activation.

NK cells may be isolated from peripheral blood mononuclear cells (PBMCs), or derived from human embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs). The primary NK cells isolated from PBMCs may be further expanded for adoptive immunotherapy. Strategies and protocols useful for the expansion of NK cells may include interleukin 2 (IL2) stimulation and the use of autologous feeder cells, or the use of genetically modified allogeneic feeder cells. In some aspects, NK cells can be selectively expanded with a combination of stimulating ligands including IL15, IL21, IL2, 41BBL, IL12, IL18, MICA, 2B4, LFA-1, and BCM1/SLAMF2 (e.g., US patent publication NO: US20150190471).

Immune cells expressing effector modules comprising a CAR and/or other immunotherapeutic agents can be used as cancer immunotherapy. The immunotherapy comprises the cells expressing a CAR and/or other immunotherapeutic agents as an active ingredient, and may further comprise a suitable excipient. Examples of the excipient may include the pharmaceutically acceptable excipients, including various cell culture media, and isotonic sodium chloride.

In some embodiments, cells of the present invention may be dendritic cells that are genetically modified to express the compositions of the invention. Such cells may be used as cancer vaccines.

III. Pharmaceutical Compositions and Formulations

The present invention further provides pharmaceutical compositions comprising one or more biocircuits, effector modules, SREs (e.g., DDs), stimuli and payloads of interest (i.e., immunotherapeutic agents), vectors, cells and other components of the invention, and optionally at least one pharmaceutically acceptable excipient or inert ingredient.

As used herein the term “pharmaceutical composition” refers to a preparation of biocircuits, SREs, stimuli and payloads of interest (i.e., immunotherapeutic agents), other components, vectors, cells and described herein, or pharmaceutically acceptable salts thereof, optionally with other chemical components such as physiologically suitable carriers and excipients. The pharmaceutical compositions of the invention comprise an effective amount of one or more active compositions of the invention. The preparation of a pharmaceutical composition that contains at least one composition of the present invention and/or an additional active ingredient will be known to those skilled in the art considering the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.

The term “excipient” or “inert ingredient” refers to an inactive substance added to a pharmaceutical composition and formulation to further facilitate administration of an active ingredient. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to any one or more biocircuits, effector modules, SREs, stimuli and payloads of interest (i.e., immunotherapeutic agents), other components, vectors, and cells to be delivered as described herein. The phrases “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.

In some embodiments, pharmaceutical compositions and formulations are administered to humans, human patients or subjects. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, non-human mammals, including agricultural animals such as cattle, horses, chickens and pigs, domestic animals such as cats, dogs, or research animals such as mice, rats, rabbits, dogs and non-human primates. It will be understood that, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

A pharmaceutical composition and formulation in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The compositions of the present invention may be formulated in any manner suitable for delivery. The formulation may be, but is not limited to, nanoparticles, poly (lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids and combinations thereof.

In one embodiment, the formulation is a nanoparticle which may comprise at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids. In another aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA and DODMA.

For polynucleotides of the invention, the formulation may be selected from any of those taught, for example, in International Application PCT/US2012/069610, the contents of which are incorporated herein by reference in its entirety.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient or inert ingredient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1 and 100, e.g., between 0.5 and 50, between 1-30, between 5-80, at least 80 (w/w) active ingredient.

Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of compositions of the present invention, “effective against” for example a cancer, indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10 in a measurable parameter of disease, and preferably at least 20, 30, 40, 50 or more can be indicative of effective treatment. Efficacy for a given composition or formulation of the present invention can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change is observed.

IV. Delivery Modalities and/or Vectors Vectors

The present invention also provides vectors that package polynucleotides of the invention encoding biocircuits, effector modules, SREs (DDs) and payload constructs, and combinations thereof. In some embodiments, polynucleotides encoding destabilizing domains, effector modules and biocircuit systems, are provided. Vectors comprising polynucleotides of the invention are provided. In some aspects, the vector may be a non-viral vector, or a viral vector. In some embodiments, the vector of the invention is a viral vector. The viral vector may include, but is not limited to a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector. In some embodiments, the vector of the invention may be a non-viral vector, such as a nanoparticles and liposomes.

Vectors of the present invention may also be used to deliver the packaged polynucleotides to a cell, a local tissue site or a subject. These vectors may be of any kind, including DNA vectors, RNA vectors, plasmids, viral vectors and particles. Viral vector technology is well known and described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses, which are useful as vectors include, but are not limited to lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes simplex viral vectors, retroviral vectors, oncolytic viruses, and the like.

In general, vectors contain an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease site, and one or more selectable markers e.g. a drug resistance gene.

As used herein a promoter is defined as a DNA sequence recognized by transcription machinery of the cell, required to initiate specific transcription of the polynucleotide sequence of the present invention. Vectors can comprise native or non-native promoters operably linked to the polynucleotides of the invention. The promoters selected may be strong, weak, constitutive, inducible, tissue specific, development stage-specific, and/or organism specific. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of polynucleotide sequence that is operatively linked to it. Another example of a preferred promoter is Elongation Growth Factor-1. Alpha (EF-1. alpha). Other constitutive promoters may also be used, including, but not limited to simian virus 40 (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV), long terminal repeat (LTR), promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter as well as human gene promoters including, but not limited to the phosphoglycerate kinase (PGK) promoter, actin promoter, the myosin promoter, the hemoglobin promoter, the Ubiquitin C (Ubc) promoter, the human U6 small nuclear protein promoter and the creatine kinase promoter. In some instances, inducible promoters such as but not limited to metallothionine promoter, glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter may be used. In some embodiments, the promoter may be selected from the following a CMV promoter, comprising a nucleotide sequence of SEQ ID NO. 335, an EF1a promoter, comprising a nucleotide sequence of SEQ ID NOs. 336-337, and a PGK promoter, comprising a nucleotide sequence of SEQ ID NO. 338.

In some embodiments, the optimal promoter may be selected based on its ability to achieve minimal expression of the SREs and payloads of the invention in the absence of the ligand and detectable expression in the presence of the ligand.

Additional promoter elements e.g. enhancers may be used to regulate the frequency of transcriptional initiation. Such regions may be located 10-100 base pairs upstream or downstream of the start site. In some instances, two or more promoter elements may be used to cooperatively or independently activate transcription.

In some embodiments, the recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell into which the vector is to be introduced.

1. Lentiviral Vectors

In some embodiments, lentiviral vectors/particles may be used as vehicles and delivery modalities. Lentiviruses are subgroup of the Retroviridae family of viruses, named because reverse transcription of viral RNA genomes to DNA is required before integration into the host genome. As such, the most important features of lentiviral vehicles/particles are the integration of their genetic material into the genome of a target/host cell. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1 and HIV-2, the Simian Immunodeficiency Virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi and caprine arthritis encephalitis virus (CAEV).

Typically, lentiviral particles making up the gene delivery vehicle are replication defective on their own (also referred to as “self-inactivating”). Lentiviruses can infect both dividing and non-dividing cells by virtue of the entry mechanism through the intact host nuclear envelope (Naldini L et al., Curr. Opin. Biotechnol, 1998, 9: 457-463). Recombinant lentiviral vehicles/particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes Env, Vif, Vpr, Vpu, Nef and Tat are deleted making the vector biologically safe. Correspondingly, lentiviral vehicles, for example, derived from HIV-1/HIV-2 can mediate the efficient delivery, integration and long-term expression of transgenes into non-dividing cells. As used herein, the term “recombinant” refers to a vector or other nucleic acid containing both lentiviral sequences and non-lentiviral retroviral sequences.

Lentiviral particles may be generated by co-expressing the virus packaging elements and the vector genome itself in a producer cell such as human HEK293T cells. These elements are usually provided in three (in second generation lentiviral systems) or four separate plasmids (in third generation lentiviral systems). The producer cells are co-transfected with plasmids that encode lentiviral components including the core (i.e. structural proteins) and enzymatic components of the virus, and the envelope protein(s) (referred to as the packaging systems), and a plasmid that encodes the genome including a foreign transgene, to be transferred to the target cell, the vehicle itself (also referred to as the transfer vector). In general, the plasmids or vectors are included in a producer cell line. The plasmids/vectors are introduced via transfection, transduction or infection into the producer cell line. Methods for transfection, transduction or infection are well known by those of skill in the art. As non-limiting example, the packaging and transfer constructs can be introduced into producer cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gln synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.

The producer cell produces recombinant viral particles that contain the foreign gene, for example, the effector module of the present invention. The recombinant viral particles are recovered from the culture media and titrated by standard methods used by those of skill in the art. The recombinant lentiviral vehicles can be used to infect target cells.

Cells that can be used to produce high-titer lentiviral particles may include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Ther., 2005, 11: 452-459), FreeStyle™ 293 Expression System (ThermoFisher, Waltham, Mass.), and other HEK293T-based producer cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22 (3):357-369; Lee et al., Biotechnol Bioeng, 2012, 10996): 1551-1560; Throm et al., Blood. 2009, 113(21): 5104-5110; the contents of each of which are incorporated herein by reference in their entirety).

In some aspects, the envelope proteins may be heterologous envelop proteins from other viruses, such as the G protein of vesicular stomatitis virus (VSV G) or baculoviral gp64 envelop proteins. The VSV-G glycoprotein may especially be chosen among species classified in the vesiculovirus genus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV) and Vesicular stomatitis New Jersey virus (VSNJV) and/or stains provisionally classified in the vesiculovirus genus as Grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAV), Kwatta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), Ulcerative disease rhabdovirus (UDRV) and Yug Bogdanovac virus (YBV). The gp64 or other baculoviral env protein can be derived from Autographa californica nucleopolyhedrovirus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fumiferana nucleopolyhedrovirus, Orgyia pseudotsugata single capsid nuclear polyhedrosis virus, Epiphyas postvittana nucleopolyhedrovirus, Hyphantria cunea nucleopolyhedrovirus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nucleopolyhedrovirus or Batken virus.

Additional elements provided in lentiviral particles may comprise retroviral LTR (long-terminal repeat) at either 5′ or 3′ terminus, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof, and a locus control region (LCR) or active portion thereof. Other elements include central polypurine tract (cPPT) sequence to improve transduction efficiency in non-dividing cells, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) which enhances the expression of the transgene, and increases titer. The effector module is linked to the vector.

Methods for generating recombinant lentiviral particles are discussed in the art, for example, U.S. Pat. NOs.: 8, 846, 385; 7,745, 179; 7,629,153; 7,575,924; 7,179, 903; and 6, 808, 905; the contents of each of which are incorporated herein by reference in their entirety.

Lentivirus vectors used may be selected from, but are not limited to pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJM1-EGFP, pULTRA, pInducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionII.

Lentiviral vehicles known in the art may also be used (See, U.S. Pat. NOs. 9, 260, 725; 9,068,199; 9,023,646; 8,900,858; 8,748,169; 8,709,799; 8,420,104; 8,329,462; 8,076,106; 6,013,516; and 5,994,136; International Patent Publication NO.: WO2012079000; the contents of each of which are incorporated herein by reference in their entirety).

2. Retroviral Vectors (γ-Retroviral Vectors)

In some embodiments, retroviral vectors may be used to package and deliver the biocircuits, biocircuit components, effector modules, SREs or payload constructs of the present invention. Retroviral vectors (RVs) allow the permanent integration of a transgene in target cells. In addition to lentiviral vectors based on complex HIV-1/2, retroviral vectors based on simple gamma-retroviruses have been widely used to deliver therapeutic genes and demonstrated clinically as one of the most efficient and powerful gene delivery systems capable of transducing a broad range of cell types. Example species of Gamma retroviruses include the murine leukemia viruses (MLVs) and the feline leukemia viruses (FeLV).

In some embodiments, gamma-retroviral vectors derived from a mammalian gamma-retrovirus such as murine leukemia viruses (MLVs), are recombinant. The MLV families of gamma retroviruses include the ecotropic, amphotropic, xenotropic and polytropic subfamilies. Ecotropic viruses are able to infect only murine cells using mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human and other species through the Pit-2 receptor. One example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xpr1) receptor, but differ in their species tropism. Xenotropic viruses such as NZB-9-1 infect human and other species but not murine species, whereas polytropic viruses such as focus-forming viruses (MCF) infect murine, human and other species.

Gamma-retroviral vectors may be produced in packaging cells by co-transfecting the cells with several plasmids including one encoding the retroviral structural and enzymatic (gag-pol) polyprotein, one encoding the envelope (env) protein, and one encoding the vector mRNA comprising polynucleotide encoding the compositions of the present invention that is to be packaged in newly formed viral particles.

In some aspects, the recombinant gamma-retroviral vectors are pseudotyped with envelope proteins from other viruses. Envelope glycoproteins are incorporated in the outer lipid layer of the viral particles which can increase/alter the cell tropism. Exemplary envelop proteins include the gibbon ape leukemia virus envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), or Simian endogenous retrovirus envelop protein, or Measles Virus H and F proteins, or Human immunodeficiency virus gp120 envelope protein, or cocal vesiculovirus envelop protein (See, e.g., U.S. application publication NO.: 2012/164118; the contents of which are incorporated herein by reference in its entirety). In other aspects, envelope glycoproteins may be genetically modified to incorporate targeting/binding ligands into gamma-retroviral vectors, binding ligands including, but not limited to, peptide ligands, single chain antibodies and growth factors (Waehler et al., Nat. Rev. Genet. 2007, 8(8):573-587; the contents of which are incorporated herein by reference in its entirety). These engineered glycoproteins can retarget vectors to cells expressing their corresponding target moieties. In other aspects, a “molecular bridge” may be introduced to direct vectors to specific cells. The molecular bridge has dual specificities: one end can recognize viral glycoproteins, and the other end can bind to the molecular determinant on the target cell. Such molecular bridges, for example ligand-receptor, avidin-biotin, and chemical conjugations, monoclonal antibodies and engineered fusogenic proteins, can direct the attachment of viral vectors to target cells for transduction (Yang et al., Biotechnol. Bioeng., 2008, 101(2): 357-368; and Maetzig et al., Viruses, 2011, 3, 677-713; the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, the recombinant gamma-retroviral vectors are self-inactivating (SIN) gammaretroviral vectors. The vectors are replication incompetent. SIN vectors may harbor a deletion within the 3′ U3 region initially comprising enhancer/promoter activity. Furthermore, the 5′ U3 region may be replaced with strong promoters (needed in the packaging cell line) derived from Cytomegalovirus or RSV, or an internal promoter of choice, and/or an enhancer element. The choice of the internal promoters may be made according to specific requirements of gene expression needed for a particular purpose of the invention.

In some embodiments, polynucleotides encoding the biocircuit, biocircuit components, effector module, SRE are inserted within the recombinant viral genome. The other components of the viral mRNA of a recombinant gamma-retroviral vector may be modified by insertion or removal of naturally occurring sequences (e.g., insertion of an IRES, insertion of a heterologous polynucleotide encoding a polypeptide or inhibitory nucleic acid of interest, shuffling of a more effective promoter from a different retrovirus or virus in place of the wild-type promoter and the like). In some examples, the recombinant gamma-retroviral vectors may comprise modified packaging signal, and/or primer binding site (PBS), and/or 5′-enhancer/promoter elements in the U3-region of the 5′-long terminal repeat (LTR), and/or 3′-SIN elements modified in the U3-region of the 3′-LTR. These modifications may increase the titers and the ability of infection.

Gamma retroviral vectors suitable for delivering biocircuit components, effector modules, SREs or payload constructs of the present invention may be selected from those disclosed in U.S. Pat. Nos. 8,828,718; 7,585,676; 7,351,585; U.S. application publication NO.: 2007/048285; PCT application publication NOs.: WO2010/113037; WO2014/121005; WO2015/056014; and EP Pat. NOs.: EP1757702; EP1757703 (the contents of each of which are incorporated herein by reference in their entirety).

3. Adeno-Associated Viral Vectors (AAV)

In some embodiments, polynucleotides of present invention may be packaged into recombinant adeno-associated viral (rAAV) vectors. Such vectors or viral particles may be designed to utilize any of the known serotype capsids or combinations of serotype capsids. The serotype capsids may include capsids from any identified AAV serotypes and variants thereof, for example, AAV1, AAV2, AAV2G9, AAV3, AAV4, AAV4-4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAVrh10.

In one embodiment, the AAV serotype may be or have a sequence as described in United States Publication No. US20030138772, herein incorporated by reference in its entirety, such as, but not limited to, AAV1 (SEQ ID NO: 6 and 64 of US20030138772), AAV2 (SEQ ID NO: 7 and 70 of US20030138772), AAV3 (SEQ ID NO: 8 and 71 of US20030138772), AAV4 (SEQ ID NO: 63 of US20030138772), AAV5 (SEQ ID NO: 114 of US20030138772), AAV6 (SEQ ID NO: 65 of US20030138772), AAV7 (SEQ ID NO: 1-3 of US20030138772), AAV8 (SEQ ID NO: 4 and 95 of US20030138772), AAV9 (SEQ ID NO: 5 and 100 of US20030138772), AAV10 (SEQ ID NO: 117 of US20030138772), AAV11 (SEQ ID NO: 118 of US20030138772), AAV12 (SEQ ID NO: 119 of US20030138772), AAVrh10 (amino acids 1 to 738 of SEQ ID NO: 81 of US20030138772) or variants thereof. Non-limiting examples of variants include SEQ ID NOs: 9, 27-45, 47-62, 66-69, 73-81, 84-94, 96, 97, 99, 101-113 of US20030138772, the contents of which are herein incorporated by reference in their entirety.

In one embodiment, the AAV serotype may have a sequence as described in Pulicherla et al. (Molecular Therapy, 2011, 19(6):1070-1078), U.S. Pat. Nos. 6,156,303; 7,198,951; U.S. Patent Publication NOs.: US2015/0159173 and US2014/0359799; and International Patent Publication NOs.: WO1998/011244, WO2005/033321 and WO2014/14422; the contents of each of which are incorporated herein by reference in their entirety.

AAV vectors include not only single stranded vectors but self-complementary AAV vectors (scAAVs). scAAV vectors contain DNA which anneals together to form double stranded vector genome. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.

The rAAV vectors may be manufactured by standard methods in the art such as by triple transfection, in sf9 insect cells or in suspension cell cultures of human cells such as HEK293 cells.

The biocircuits, biocircuit components, effector modules, SREs or payload constructs may be encoded in one or more viral genomes to be packaged in the AAV capsids taught herein.

Such vectors or viral genomes may also include, in addition to at least one or two ITRs (inverted terminal repeats), certain regulatory elements necessary for expression from the vector or viral genome. Such regulatory elements are well known in the art and include for example promoters, introns, spacers, stuffer sequences, and the like.

In some embodiments, more than one effector module or SRE (e.g. DD) may be encoded in a viral genome.

4. Oncolytic Viral Vector

In some embodiments, polynucleotides of present invention may be packaged into oncolytic viruses, such as vaccine viruses. Oncolytic vaccine viruses may include viral particles of a thymidine kinase (TK)-deficient, granulocyte macrophage (GM)-colony stimulating factor (CSF)-expressing, replication-competent vaccinia virus vector sufficient to induce oncolysis of cells in the tumor (e.g., U.S. Pat. No. 9,226,977).

In some embodiments, the viral vector of the invention may comprise two or more immunotherapeutic agents taught herein, wherein the two or more immunotherapeutic agents may be included in one effector module under the regulation of the same DD. In this case, the two or more immunotherapeutic agents are tuned by the same stimulus simultaneously. In other embodiments, the viral vector of the invention may comprise two or more effector modules, wherein each effector module comprises a different immunotherapeutic agent. In this case, the two or more effector modules and immunotherapeutic agents are tuned by different stimuli, providing separately independent regulation of the two or more components.

5. Messenger RNA (mRNA)

In some embodiments, the effector modules of the invention may be designed as a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. Such mRNA molecules may have the structural components or features of any of those taught in International Application number PCT/US2013/030062, the contents of which are incorporated herein by reference in its entirety.

Polynucleotides of the invention may also be designed as taught in, for example, Ribostem Limited in United Kingdom patent application serial number 0316089.2 filed on Jul. 9, 2003 now abandoned, PCT application number PCT/GB2004/002981 filed on Jul. 9, 2004 published as WO2005005622, United States patent application national phase entry serial number 10/563,897 filed on Jun. 8, 2006 published as US20060247195 now abandoned, and European patent application national phase entry serial number EP2004743322 filed on Jul. 9, 2004 published as EP1646714 now withdrawn; Novozymes, Inc. in PCT application number PCT/US2007/88060 filed on Dec. 19, 2007 published as WO2008140615, United States patent application national phase entry serial number 12/520,072 filed on Jul. 2, 2009 published as US20100028943 and European patent application national phase entry serial number EP2007874376 filed on Jul. 7, 2009 published as EP2104739; University of Rochester in PCT application number PCT/US2006/46120 filed on Dec. 4, 2006 published as WO2007064952 and U.S. patent application Ser. No. 11/606,995 filed on Dec. 1, 2006 published as US20070141030; BioNTech AG in European patent application serial number EP2007024312 filed Dec. 14, 2007 now abandoned, PCT application number PCT/EP2008/01059 filed on Dec. 12, 2008 published as WO2009077134, European patent application national phase entry serial number EP2008861423 filed on Jun. 2, 2010 published as EP2240572, United States patent application national phase entry serial number 12/735,060 filed Nov. 24, 2010 published as US20110065103, German patent application serial number DE 10 2005 046 490 filed Sep. 28, 2005, PCT application PCT/EP2006/0448 filed Sep. 28, 2006 published as WO2007036366, national phase European patent EP1934345 published Mar. 21, 2012 and national phase U.S. patent application Ser. No. 11/992,638 filed Aug. 14, 2009 published as 20100129877; Immune Disease Institute Inc. in U.S. patent application Ser. No. 13/088,009 filed Apr. 15, 2011 published as US20120046346 and PCT application PCT/US2011/32679 filed Apr. 15, 2011 published as WO20110130624; Shire Human Genetic Therapeutics in U.S. patent application Ser. No. 12/957,340 filed on Nov. 20, 2010 published as US20110244026; Sequitur Inc. in PCT application PCT/US1998/019492 filed on Sep. 18, 1998 published as WO1999014346; The Scripps Research Institute in PCT application number PCT/US2010/00567 filed on Feb. 24, 2010 published as WO2010098861, and United States patent application national phase entry serial number 13/203,229 filed Nov. 3, 2011 published as US20120053333; Ludwig-Maximillians University in PCT application number PCT/EP2010/004681 filed on Jul. 30, 2010 published as WO2011012316; Cellscript Inc. in U.S. Pat. No. 8,039,214 filed Jun. 30, 2008 and granted Oct. 18, 2011, U.S. patent application Ser. No. 12/962,498 filed on Dec. 7, 2010 published as US20110143436, 12/962,468 filed on Dec. 7, 2010 published as US20110143397, 13/237,451 filed on Sep. 20, 2011 published as US20120009649, and PCT applications PCT/US2010/59305 filed Dec. 7, 2010 published as WO2011071931 and PCT/US2010/59317 filed on Dec. 7, 2010 published as WO2011071936; The Trustees of the University of Pennsylvania in PCT application number PCT/US2006/32372 filed on Aug. 21, 2006 published as WO2007024708, and United States patent application national phase entry serial number 11/990,646 filed on Mar. 27, 2009 published as US20090286852; Curevac GMBH in German patent application serial numbers DE10 2001 027 283.9 filed Jun. 5, 2001, DE10 2001 062 480.8 filed Dec. 19, 2001, and DE 20 2006 051 516 filed Oct. 31, 2006 all abandoned, European patent numbers EP1392341 granted Mar. 30, 2005 and EP1458410 granted Jan. 2, 2008, PCT application numbers PCT/EP2002/06180 filed Jun. 5, 2002 published as WO2002098443, PCT/EP2002/14577 filed on Dec. 19, 2002 published as WO2003051401, PCT/EP2007/09469 filed on Dec. 31, 2007 published as WO2008052770, PCT/EP2008/03033 filed on Apr. 16, 2008 published as WO2009127230, PCT/EP2006/004784 filed on May 19, 2005 published as WO2006122828, PCT/EP2008/00081 filed on Jan. 9, 2007 published as WO2008083949, and U.S. patent application Ser. No. 10/729,830 filed on Dec. 5, 2003 published as US20050032730, 10/870,110 filed on Jun. 18, 2004 published as US20050059624, 11/914,945 filed on Jul. 7, 2008 published as US20080267873, 12/446,912 filed on Oct. 27, 2009 published as US2010047261 now abandoned, 12/522,214 filed on Jan. 4, 2010 published as US20100189729, 12/787,566 filed on May 26, 2010 published as US20110077287, 12/787,755 filed on May 26, 2010 published as US20100239608, 13/185,119 filed on Jul. 18, 2011 published as US20110269950, and 13/106,548 filed on May 12, 2011 published as US20110311472 all of which are herein incorporated by reference in their entirety.

In some embodiments, the effector modules may be designed as self-amplifying RNA. “Self-amplifying RNA” as used herein refers to RNA molecules that can replicate in the host resulting in the increase in the amount of the RNA and the protein encoded by the RNA. Such self-amplifying RNA may have structural features or components of any of those taught in International Patent Application Publication No. WO2011005799 (the contents of which are incorporated herein by reference in their entirety)

V. Dosing, Delivery and Administrations

The compositions of the invention may be delivered to a cell or a subject through one or more routes and modalities. The viral vectors containing one or more effector modules, SREs, immunotherapeutic agents and other components described herein may be used to deliver them to a cell and/or a subject. Other modalities may also be used such as mRNAs, plasmids, and as recombinant proteins.

1. Delivery to Cells

In another aspect of the invention, polynucleotides encoding biocircuits, effector modules, SREs (e.g., DDs), payloads of interest (immunotherapeutic agents) and compositions of the invention and vectors comprising said polynucleotides may be introduced into cells such as immune effector cells.

In one aspect of the invention, polynucleotides encoding biocircuits, effector modules, SREs (e.g., DDs), payloads of interest (immunotherapeutic agents) and compositions of the invention, may be packaged into viral vectors or integrated into viral genomes allowing transient or stable expression of the polynucleotides. Preferable viral vectors are retroviral vectors including lentiviral vectors. In order to construct a retroviral vector, a polynucleotide molecule encoding a biocircuit, an effector module, a DD or a payload of interest (i.e. an immunotherapeutic agent) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes, but without the LTR and packaging components. The recombinant retroviral particles are secreted into the culture media, then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are especially preferred as they are capable of infecting both dividing and non-dividing cells.

Vectors may also be transferred to cells by non-viral methods by physical methods such as needles, electroporation, sonoporation, hyrdoporation; chemical carriers such as inorganic particles (e.g. calcium phosphate, silica, gold) and/or chemical methods. In some embodiments, synthetic or natural biodegradable agents may be used for delivery such as cationic lipids, lipid nano emulsions, nanoparticles, peptide based vectors, or polymer based vectors.

In some embodiments, the polypeptides of the invention may be delivered to the cell directly. In one embodiment, the polypeptides of the invention may be delivered using synthetic peptides comprising an endosomal leakage domain (ELD) fused to a cell penetration domain (CLD). The polypeptides of the invention are co introduced into the cell with the ELD-CLD-synthetic peptide. ELDs facilitate the escape of proteins that are trapped in the endosome, into the cytosol. Such domains are derived proteins of microbial and viral origin and have been described in the art. CPDs allow the transport of proteins across the plasma membrane and have also been described in the art. The ELD-CLD fusion proteins synergistically increase the transduction efficiency when compared to the co-transduction with either domain alone. In some embodiments, a histidine rich domain may optionally be added to the shuttle construct as an additional method of allowing the escape of the cargo from the endosome into the cytosol. The shuttle may also include a cysteine residue at the N or C terminus to generate multimers of the fusion peptide. Multimers of the ELD-CLD fusion peptides generated by the addition of cysteine residue to the terminus of the peptide show even greater transduction efficiency when compared to the single fusion peptide constructs. The polypeptides of the invention may also be appended to appropriate localization signals to direct the cargo to the appropriate sub-cellular location e.g. nucleus. In some embodiments any of the ELDs, CLDs or the fusion ELD-CLD synthetic peptides taught in the International Patent Publication, WO2016161516 and WO2017175072 may be useful in the present invention (the contents of each of which are herein incorporated by reference in their entirety).

2. Dosing

The present invention provides methods comprising administering any one or more compositions for immunotherapy to a subject in need thereof. These may be administered to a subject using any amount and any route of administration effective for preventing or treating a clinical condition such as cancer, infection diseases and other immunodeficient diseases.

Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, or prophylactically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, previous or concurrent therapeutic interventions and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

Compositions of the invention may be used in varying doses to avoid T cell anergy, prevent cytokine release syndrome and minimize toxicity associated with immunotherapy. For example, low doses of the compositions of the present invention may be used to initially treat patients with high tumor burden, while patients with low tumor burden may be treated with high and repeated doses of the compositions of the invention to ensure recognition of a minimal tumor antigen load. In another instance, the compositions of the present invention may be delivered in a pulsatile fashion to reduce tonic T cell signaling and enhance persistence in vivo. In some aspects, toxicity may be minimized by initially using low doses of the compositions of the invention, prior to administering high doses. Dosing may be modified if serum markers such as ferritin, serum C-reactive protein, IL6, IFN γ, and TNF-α are elevated.

In one embodiment, polypeptides and/or polynucleotides expressing the compositions described herein e.g. effector modules, are administered to a subject in need thereof to treat cancer. In one embodiment, a population of cells comprising the biocircuits, effector modules and/or the SREs described herein is administered to a subject in need thereof to treat cancer.

In one embodiment, the cells expressing the compositions described herein is administered at a dose and/or dosing schedule described herein.

In one embodiment, compositions are introduced into immune cells (e.g., T cells, NK cells). The compositions may be introduced into the immune cells by methods including but not limited to viral transduction, transfection and/or in vitro transcription. In some embodiments, the immune cells are transduced with retroviruses. In one aspect, the retrovirus may be a lentivirus. In some aspects, the titer of the lentiviruses may be used to tune the expression of the payload. In some embodiments, the titer of the lentivirus may have a multiplicity of infection (MOI) ranging from 0.001-0.01, 0.01-0.1, 0.1-1, or 1-10. In some embodiments, the MOI may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the subject (e.g., human) receives an initial administration of immune cells comprising the compositions described herein e.g. SREs, and one or more subsequent administrations of cells. In some embodiments, a therapeutically effective amount of the compositions and/or cells described herein may be administered to the subject. In some embodiments, the therapeutically effective amount may indicate the precise amount required to tumor inhibition, tumor prevalence and/or tumor burden. The therapeutically effective amount may be determined with consideration of the subject's age, weight, tumor size, sex, extent of infection or metastasis. In some embodiments, the cells expressing the compositions described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight of the subject. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988; the contents of which are incorporated herein by reference in their entirety).

In some aspects, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells. These T cell isolates may be expanded by methods known in the art and treated such that the compositions of the invention may be introduced into the cell. Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the immune cells of the present invention. In an additional aspect, expanded cells are administered before or following surgery.

The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.

In one embodiment, the compositions are introduced into immune cells (e.g., T cells, NK cells), e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of immune cells (e.g., T cells, NK cells) of the invention, and one or more subsequent administrations of the immune cells (e.g., T cells, NK cells) of the invention, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the immune cells (e.g., T cells, NK cells) of the invention are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the immune cells (e.g., T cells, NK cells) of the invention are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of the immune cells (e.g., T cells, NK cells) per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no immune cells (e.g., T cells, NK cells) administrations, and then one or more additional administration of the immune cells (e.g., T cells, NK cells) (e.g., more than one administration of the immune cells (e.g., T cells, NK cells) per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of immune cells (e.g., T cells, NK cells), and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the immune cells (e.g., T cells, NK cells) are administered every other day for 3 administrations per week. In one embodiment, the immune cells (e.g., T cells, NK cells) of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks. In some embodiments, a dose of immune cells expressing compositions of the invention described herein comprises about 1×10⁶, 1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸ 3×10⁸, or 5×10⁸ cells/kg. In some embodiments, a dose of immune cells comprises at least about 1×10⁶, 1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, or 5×10⁸ cells/kg. In some embodiments, a dose of immune cells comprises up to about 1×10⁶, 1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, or 5×10⁸ cells/kg. In some embodiments, a dose of immune cells comprises about 1.1×10⁶, 1.8×10⁷ cells/kg. In some embodiments, a dose of immune cells comprises about 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells. In some embodiments, a dose of the immune cells comprises at least about 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells. In some embodiments, a dose of immune cells comprises up to about 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells. In some embodiments, a dose of the immune cells comprises up to about 1×10⁷, 1.5×10⁷, 2×10⁷, 2.5×10⁷, 3×10⁷, 3.5×10⁷, 4×10⁷, 5×10⁷, 1×10⁸, 1.5×10⁸, 2×10⁸, 2.5×10⁸, 3×10⁸, 3.5×10⁸, 4×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells. In some embodiments, a dose of immune cells comprises up to about 1-3×10⁷ to 1-3×10⁸ cells.

In one embodiment, the cells expressing the compositions described herein, are administered as a first line treatment for the disease, e.g., the cancer, e.g., the cancer described herein. In another embodiment, the cells expressing the compositions described herein, are administered as a second, third, fourth line treatment for the disease, e.g., the cancer, e.g., the cancer described herein. In some embodiments, the subject may undergo preconditioning prior to the administration of the cells.

Also provided herein are methods of administering ligands in accordance with the invention to a subject in need thereof. The ligand may be administered to a subject or to cells, using any amount and any route of administration effective for tuning the biocircuits of the invention. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, a mammal, or an animal. Compositions in accordance with the invention are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. In certain embodiments, the ligands in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 100 mg/kg, from about 50 mg/kg to about 500 mg/kg, from about 100 mg/kg to about 1000 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired effect. In some embodiments, the dosage levels may be 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 100 mg/kg, 110 mg/kg, 120 mg/kg, 130 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 170 mg/kg, 180 mg/kg, 190 mg/kg or mg/kg of subject body weight per day, or more times a day, to obtain the desired effect.

The present disclosure provides methods for delivering to a cell or tissue any of the ligands described herein, comprising contacting the cell or tissue with said ligand and can be accomplished in vitro, ex vivo, or in vivo. In certain embodiments, the ligands in accordance with the present invention may be administered to cells at dosage levels sufficient to deliver from about 1 nM to about 10 nM, from about 5 nM to about 50 nM, from about 10 nM to about 100 nM, from about 50 nM to about 500 nM, from about 100 nM to about 1000 nM, from about 1 μM to about 10 μM, from about 504 to about 50 μM, from about 10 μM to about 100 μM from about 2504 to about 250 μM, from about 50 μM to about 500 μM. In some embodiments, the ligand may be administered to cells at doses selected from but not limited to 0.006404, 0.003204, 0.01604, 0.0804, 2 μl, M, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 250 μM.

The desired dosage of the ligands of the present invention may be delivered only once, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. As used herein, a “split dose” is the division of “single unit dose” or total daily dose into two or more doses, e.g., two or more administrations of the “single unit dose”. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event. The desired dosage of the ligand of the present invention may be administered as a “pulse dose” or as a “continuous flow”. As used herein, a “pulse dose” is a series of single unit doses of any therapeutic administered with a set frequency over a period of time. As used herein, a “continuous flow” is a dose of therapeutic administered continuously for a period of time in a single route/single point of contact, i.e., continuous administration event. A total daily dose, an amount given or prescribed in 24-hour period, may be administered by any of these methods, or as a combination of these methods, or by any other methods suitable for a pharmaceutical administration.

3. Administration

In some embodiments, the compositions for immunotherapy may be administered to cells ex vivo and subsequently administered to the subject. Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating cytotoxic T cells are described in U.S. Pat. Nos. 6,805,861 and 6,531,451; the contents of each of which are incorporated herein by reference in their entirety. Isolation of NK cells is described in U.S. Pat. NOs.: 7,435, 596; the contents of which are incorporated by reference herein in its entirety.

In some embodiments, depending upon the nature of the cells, the cells may be introduced into a host organism e.g. a mammal, in a wide variety of ways including by injection, transfusion, infusion, local instillation or implantation. In some aspects, the cells of the invention may be introduced at the site of the tumor. The number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, or the like. The cells may be in a physiologically-acceptable medium.

In some embodiments, the cells of the invention may be administrated in multiple doses to subjects having a disease or condition. The administrations generally effect an improvement in one or more symptoms of cancer or a clinical condition and/or treat or prevent cancer or clinical condition or symptom thereof.

In some embodiments, the compositions for immunotherapy may be administered in vivo. In some embodiments, polypeptides of the present invention comprising biocircuits, effector molecules, SREs, payloads of interest (immunotherapeutic agents) and compositions of the invention may be delivered in vivo to the subject. In vivo delivery of immunotherapeutic agents is well described in the art. For example, methods of delivery of cytokines are described in the E. P. Pat. No.: EP0930892 A1, the contents of which are incorporated herein by reference.

Routes of Delivery

The pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs (e.g., DDs), payloads (i.e. immunotherapeutic agents), vectors and cells of the present invention may be administered by any route to achieve a therapeutically effective outcome.

These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intra-arterial (into an artery), intramuscular (into a muscle), intracranial (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intrasinal infusion, intravitreal, (through the eye), intravenous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal.

Kits

The also provides a kit comprising any of the polynucleotides or expression vectors described herein.

The present invention includes a variety of kits for conveniently and/or effectively carrying out methods of the present invention. Typically, kits will comprise sufficient amounts and/or numbers of components to allow a user to perform one or multiple treatments of a subject(s) and/or to perform one or multiple experiments.

In one embodiment, the present invention provides kits for inhibiting genes in vitro or in vivo, comprising a biocircuit of the present invention or a combination of biocircuits of the present invention, optionally in combination with any other suitable active agents.

The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise, for example, saline, a buffered solution.

In additional embodiments, assay screening kits are provided. The kit includes a container for the screening assay. An instruction for the use of the assay and the information about the screening method are to be included in the kit.

In some embodiments, the DDs, effector modules and biocircuit system and compositions of the invention may be used as research tools to investigate protein activity in a biological system such a cell and a subject. In other embodiments, the DDs, effector modules and biocircuit system and compositions of the invention may be used for treating a disease such as a cancer and a genetic disorder.

VI. Applications

In one aspect of the present invention, methods for reducing a tumor volume or burden are provided. The methods comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising at least one biocircuit system, effector module, DD, and/or payload of interest (i.e., an immunotherapeutic agent), at least one vector, or cells to a subject having a tumor. The biocircuit system and effector module having any immunotherapeutic agent as described herein may be in forms of a polypeptide, or a polynucleotide such as mRNA, or a viral vector comprising the polynucleotide, or a cell modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).

In another aspect of the present invention, methods for inducing an anti-tumor immune response in a subject are provided. The methods comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising at least one biocircuit system, effector module, DD, and/or payload of interest (i.e., an immunotherapeutic agent), at least one vector, or cells to a subject having a tumor. The biocircuit and effector module having any immunotherapeutic agent as described herein may be in forms of a polypeptide, or a polynucleotide such as mRNA, or a viral vector comprising the polynucleotide, or a cell modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).

The methods, per the present invention, may be adoptive cell transfer (ACT) using genetically engineered cells such as immune effector cells of the invention, cancer vaccines comprising biocircuit systems, effector modules, DDs, payloads of interest (i.e., immunotherapeutic agents) of the invention, or compositions that manipulate the tumor immunosuppressive microenvironment, or the combination thereof. These treatments may be further employed with other cancer treatment such as chemotherapy and radiotherapy.

1. Adoptive Cell Transfer (Adoptive Immunotherapy)

Recent progress in the field of cancer immunology has allowed the development of several approaches to help the immune system keep the cancer at bay. Such immunotherapy approaches include the targeting of cancer antigens through monoclonal antibodies or through adoptive transfer of ex vivo engineered T cells (e.g., which contain chimeric antigen receptors or engineered T cell receptors). The present invention also provides methods for inducing immune responses in a subject using the compositions of the invention. Also provided are methods for reducing a tumor burden in a subject using the compositions of the invention. The present invention also provides immune cells engineered to include one or more polypeptides, polynucleotides, or vectors of the present invention. The cells may be immune effector cells, including T cells such as cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK T cells, cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), and tumor infiltrating lymphocytes (TILs). The engineered cell may be used for adoptive cell transfer for treating a disease (e.g., a cancer).

In some embodiments, pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention may be used in the modulation or alteration or exploitation of the immune system to target one or more cancers. This approach may also be considered with other such biological approaches, e.g., immune response modifying therapies such as the administration of interferons, interleukins, colony-stimulating factors, other monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents are also envisioned as anti-cancer therapies to be combined with the pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention.

Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the patient's own immune system to fight the cancer. In some embodiments, pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention are designed as immune-oncology therapeutics.

In some embodiments, cells which are genetically modified to express at least one biocircuit system, effector module, DD, and/or payload of interest (immunotherapeutic agent) may be used for adoptive cell therapy (ACT). As used herein, Adoptive cell transfer refers to the administration of immune cells (from autologous, allogenic or genetically modified hosts) with direct anticancer activity. ACT has shown promise in clinical application against malignant and infectious disease. For example, T cells genetically engineered to recognize CD19 have been used to treat follicular B cell lymphoma (Kochenderfer et al., Blood, 2010, 116:4099-4102; and Kochenderfer and Rosenberg, Nat Rev Clin Oncol., 2013, 10(5): 267-276) and ACT using autologous lymphocytes genetically-modified to express anti-tumor T cell receptors has been used to treat metastatic melanoma (Rosenberg and Dudley, Curr. Opin. Immunol. 2009, 21: 233-240).

Immune cells for adoptive cell therapy may be selected from but not limited to selected from a CD8+ T cell, a CD4+ T cell, a helper T cell, a natural killer (NK) cell, a NKT cell, a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), a memory T cell, a regulatory T (Treg) cell, a cytokine-induced killer (CIK) cell, a dendritic cell, a human embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof.

There are several types of cellular immunotherapies, including tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells bearing chimeric antigen receptors (CARs), and recombinant TCR technology. In some embodiments, the compositions of the present invention may be utilized to alter TIL (tumor infiltrating lymphocyte) populations in a subject. In one embodiment, any of the payloads described herein may be utilized to change the ratio of CD4 positive cells to CD8 positive populations. In some embodiments, TILs may be sorted ex vivo and engineered to express any of the cytokines described herein. Payloads of the invention may be used to expand CD4 and/or CD8 populations of TILs to enhance TIL mediated immune response.

In one embodiment, the chimeric antigen receptor (CAR) of the present invention may be a conditionally active CAR. A wild type protein or domain thereof, such as those described herein may be used to generate a conditionally active biologic protein which are reversibly or irreversibly inactivated at the wild type normal physiological conditions as well as to such conditionally active biologic proteins and domains and uses of such conditional active biologic proteins and domains are provided. Such methods and conditionally active proteins are taught in, for example, International Publication No. WO2016033331, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the CAR comprises at least one antigen specific targeting region evolved from a wild type protein or a domain thereof and one or more of a decrease in activity in the assay at the normal physiological condition compared to the antigen specific targeting region of the wild-type protein or a domain thereof, and an increase in activity in the assay under the aberrant condition compared to the antigen specific targeting region of the wild-type protein or a domain thereof.

According to the present invention, the biocircuits and systems may be used in the development and implementation of cell therapies such as adoptive cell therapy. Certain effector modules useful in cell therapy are given in FIGS. 7-12 . The biocircuits, their components, effector modules and their SREs and payloads may be used in cell therapies to effect TCR removal-TCR gene disruption, TCR engineering, to regulate epitope tagged receptors, in APC platforms for stimulating T cells, as a tool to enhance ex vivo APC stimulation, to improve methods of T cell expansion, in ex vivo stimulation with antigen, in TCR/CAR combinations, in the manipulation or regulation of TILs, in allogeneic cell therapy, in combination T cell therapy with other treatment lines (e.g. radiation, cytokines), to encode engineered TCRs, or modified TCRs, or to enhance T cells other than TCRs (e.g. by introducing cytokine genes, genes for the checkpoint inhibitors PD1, CTLA4).

In some embodiments, improved response rates are obtained in support of cell therapies.

Expansion and persistence of cell populations may be achieved through regulation or fine tuning of the payloads, e.g., the receptors or pathway components in T cells, NK cells or other immune-related cells. In some embodiments, biocircuits, their components, SREs or effector modules are designed to spatially and/or temporally control the expression of proteins which enhance T-cell or NK cell response. In some embodiments, biocircuits, their components, SREs or effector modules are designed to spatially and/or temporally control the expression of proteins which inhibit T-cell or NK cell response.

Provided herein are methods for use in adoptive cell therapy. The methods involve preconditioning a subject in need thereof, modulating immune cells with SRE, biocircuits and compositions of the present invention, administering to a subject, engineered immune cells expressing compositions of the invention and the successful engraftment of engineered cells within the subject.

In some embodiments, SREs, biocircuits and compositions of the present invention may be used to minimize preconditioning regimens associated with adoptive cell therapy. As used herein “preconditioning” refers to any therapeutic regimen administered to a subject in order to improve the outcome of adoptive cell therapy. Preconditioning strategies include, but are not limited to total body irradiation and/or lymphodepleting chemotherapy. Adoptive therapy clinical trials without preconditioning have failed to demonstrate any clinical benefit, indicating its importance in ACT. Yet, preconditioning is associated with significant toxicity and limits the subject cohort that is suitable for ACT. In some instances, immune cells for ACT may be engineered to express cytokines such as IL12 and IL15 as payload using SREs of the present invention to reduce the need for preconditioning (Pengram et al. (2012) Blood 119 (18): 4133-41; the contents of which are incorporated by reference in their entirety).

In some embodiments, the neurotoxicity may be associated with CAR or TIL therapy. Such neurotoxicity may be associated CD19-CARs. Toxicity may be due to excessive T cell infiltration into the brain. In some embodiments, neurotoxicity may be alleviated by preventing the passage of T cells through the blood brain barrier. This can be achieved by the targeted gene deletion of the endogenous alpha-4 integrin inhibitors such as tysabri/natalizumab may also be useful in the present invention.

In some embodiments, the effector modules may encode one or more cytokines. In some embodiments, the cytokine is IL15. Effector modules encoding IL15 may be designed to induce proliferation in cytotoxic populations and avoid stimulation of T regs. In other cases, the effector modules which induce proliferation in cytotoxic populations may also stimulate NK and NKT cells.

In some embodiments, effector modules may encode, or be tuned or induced to produce, one or more cytokines for expansion of cells in the biocircuits of the invention. In such cases the cells may be tested for actual expansion. Expansion may be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

In some embodiments, the tumor microenvironment may be remodeled using a biocircuit containing an effector module encoding IL17.

In some embodiments, biocircuits, their components, SREs or effector modules are designed to modulate Tregs to attenuate autoimmune disorders. In such a case, IL2 may be regulated using a singly tuned module or one having multiple tuned features as described herein.

In some embodiments, immune cells for ACT may be dendritic cells, T cells such as CD8⁺ T cells and CD4⁺ T cells, natural killer (NK) cells, NK T cells, Cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), lymphokine activated killer (LAK) cells, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (CIK) cells, and any combination thereof. In other embodiments, immune stimulatory cells for ACT may be generated from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC). In some embodiments, autologous or allogeneic immune cells are used for ACT.

In some embodiments, cells used for ACT may be T cells engineered to express T cell receptors (TCRs) with new specificities or CARs comprising an antigen-binding domain specific to an antigen on tumor cells of interest. In other embodiments, cells used for ACT may be NK cells engineered to express CARs comprising an antigen-binding domain specific to an antigen on tumor cells of interest. In addition to adoptive transfer of genetically modified T cells (e.g., CAR T cells) for immunotherapy, alternate types of CAR-expressing leukocytes, either alone, or in combination with CAR T cells may be used for adoptive immunotherapy. In one example, a mixture of T cells and NK cells may be used for ACT. The expression level of CARs in T cells and NK cells, according to the present invention, is tuned and controlled by a small molecule that binds to the DD(s) operably linked to the CAR in the effector module.

In some embodiments, NK cells engineered to express the present compositions may be used for ACT. NK cell activation induces perforin/granzyme-dependent apoptosis in target cells. NK cell activation also induces cytokine secretion such as IFN γ, TNF-α and GM-CSF. These cytokines enhance the phagocytic function of macrophages and their antimicrobial activity, and augment the adaptive immune response via up-regulation of antigen presentation by antigen presenting cells such as dendritic cells (DCs) (Reviewed by Vivier et al., Nat. Immunol., 2008, 9(5): 503-510).

Other examples of genetic modification may include the introduction of chimeric antigen receptors (CARs) and the down-regulation of inhibitory NK cell receptors such as NKG2A. Examples of CARs include, but are not limited to, CD19 and CD20 specific CARs against B cell malignancies, CARs targeting CD33 on leukemia cells, CS1 CAR and CD138 CAR on myeloma cells, GD2 CAR on neuroblastoma cells, Her2/Neu and erbB2 on breast cancer cells, carcinoembryonic antigen (CEA) on colon cancers, EpCAM on epithelial tumors, GPA7 on melanoma, NKG2D ligand on leukemia and solid tumors, and TRAIL R1 on various tumor targets. The CARs may be POIs of the effector modules and regulated by the binding of the DD with its corresponding ligand.

In another example, NK cells for ACT may be modified to express an effector module comprising the high-affinity CD16-158V polymorphism (HA-CD16) which augments NK cell mediated antibody dependent cell cytotoxicity (ADCC) against tumors. Infusion of NK cells genetically modified to express HA-CD16 could be used as a strategy to improve the efficacy of antibody-based therapies for cancer patients.

NK cells may also be genetically reprogrammed to circumvent NK cell inhibitory signals upon interaction with tumor cells. For example, using CRISPR, ZFN, or TALEN to genetically modify NK cells to silence their inhibitory receptors may enhance the anti-tumor capacity of NK cells.

Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating and expanding cytotoxic T cells are described in U.S. Pat. Nos. 6,805,861 and 6,531,451; US Patent Publication No.: US20160348072A1 and International Patent Publication NO: WO2016168595A1; the contents of each of which are incorporated herein by reference in their entirety. Isolation and expansion of NK cells is described in US Patent Publication NO.: US20150152387A1, U.S. Pat. No. 7,435,596; and Oyer, J. L. (2016). Cytotherapy. 18(5):653-63; the contents of each of which are incorporated by reference herein in its entirety. Specifically, human primary NK cells may be expanded in the presence of feeder cells e.g. a myeloid cell line that has been genetically modified to express membrane bound IL15, IL21, IL12 and 4-1BBL.

In some instances, sub populations of immune cells may be enriched for ACT. Methods for immune cell enrichment are taught in International Patent Publication NO.: WO2015039100A1. In another example, T cells positive for B and T lymphocyte attenuator marker BTLA) may be used to enrich for T cells that are anti-cancer reactive as described in U.S. Pat. No. 9,512,401 (the content of each of which are incorporated herein by reference in their entirety).

In some embodiments, immune cells for ACT may be depleted of select sub populations to enhance T cell expansion. For example, immune cells may be depleted of Foxp3+T lymphocytes to minimize the ant-tumor immune response using methods taught in US Patent Publication NO.: US 20160298081A1; the contents of which are incorporated by reference herein in their entirety.

In some embodiments, activation and expansion of T cells for ACT is achieved antigenic stimulation of a transiently expressed Chimeric Antigen Receptor (CAR) on the cell surface. Such activation methods are taught in International Patent NO.: WO2017015427, the content of which are incorporated herein by reference in their entirety.

In some embodiments, immune cells may be activated by antigens associated with antigen presenting cells (APCs). In some embodiments, the APCs may be dendritic cells, macrophages or B cells that antigen specific or nonspecific. The APCs may autologous or homologous in their organ. In some embodiments the APCs may be artificial antigen presenting cells (aAPCs) such as cell based aAPCs or acellular aAPCs. Cell based aAPCs are may be selected from either genetically modified allogeneic cells such as human erythroleukemia cells or xenogeneic cells such as murine fibroblasts and Drosophila cells. Alternatively, the APCs maybe be acellular wherein the antigens or costimulatory domains are presented on synthetic surfaces such as latex beads, polystyrene beads, lipid vesicles or exosomes.

In some embodiments, adoptive cell therapy is carried out by autologous transfer, wherein the cells are derived from a subject in need of a treatment and the cells, following isolation and processing are administered to the same subject. In other instances, ACT may involve allogenic transfer wherein the cells are isolated and/or prepared from a donor subject other than the recipient subject who ultimately receives cell therapy. The donor and recipient subject may be genetically identical, or similar or may express the same HLA class or subtype.

In some embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by the same biocircuit system. In one example, a cytokine such as IL12 and a CAR construct such as CD19 CAR are linked to the same hDHFR destabilizing domain. The expression of IL12 and CD19 CAR is tuned using TMP simultaneously. In other embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by different biocircuit systems. In one example, a cytokine such as IL12 and a CAR construct such as CD19 CAR are linked to different DDs in two separate effector modules, thereby can be tuned separately using different stimuli. In another example, a suicide gene and a CAR construct may be linked to two separate effector modules.

Following genetic modulation using SREs, biocircuits and compositions of the invention, cells are administered to the subject in need thereof. Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338; the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, immune cells for ACT may be modified to express one or more immunotherapeutic agents which facilitate immune cells activation, infiltration, expansion, survival and anti-tumor functions. The immunotherapeutic agents may be a second CAR or TCR specific to a different target molecule; a cytokine or a cytokine receptor; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; or a safety switch gene (e.g., a suicide gene) that kills activated T cells when a severe event is observed after adoptive cell transfer or when the transferred immune cells are no-longer needed.

In some embodiments, immune cells used for adoptive cell transfer can be genetically manipulated to improve their persistence, cytotoxicity, tumor targeting capacity, and ability to home to disease sites in vivo, with the overall aim of further improving upon their capacity to kill tumors in cancer patients. One example is to introduce effector modules of the invention comprising cytokines such as gamma-cytokines (IL2 and IL15) into immune cells to promote immune cell proliferation and survival. Transduction of cytokine genes (e.g., gamma-cytokines IL2 and IL15) into cells will be able to propagate immune cells without addition of exogenous cytokines and cytokine expressing NK cells have enhanced tumor cytotoxicity.

Another example includes the use of genetic modification for directing the infused NK cells to proper tumor tissues. NK cells may be genetically engineered with an effector module that encodes the CCR7 receptor to improve their migration toward one of its ligands CCL19. Other strategies may involve utilizing chemokine receptors, such as CXCR3 to improve NK cell migration to inflamed tissues, such as those infiltrated with metastatic tumors.

NK cells may be modified to become insensitive to suppressive cytokines such as TGF-β, thereby preserving their cytotoxicity. For example, NK cells can be genetically modified to express the dominant negative mutant form of TGF-β type II receptor (DNTβRII) on their surface that render NK cells resistant to the suppressive effects of TGF-β.

In some embodiments, biocircuits, their components, SREs or effector modules are designed to be significantly less immunogenic than other biocircuits or switches in the art.

As used herein, “significantly less immunogenic” refers to a detectable decrease in immunogenicity. In another embodiment, the term refers to a fold decrease in immunogenicity. In another embodiment, the term refers to a decrease such that an effective amount of the biocircuits, their components, SREs or effector modules which can be administered without triggering a detectable immune response. In another embodiment, the term refers to a decrease such that the biocircuits, their components, SREs or effector modules can be repeatedly administered without eliciting an immune response. In another embodiment, the decrease is such that the biocircuits, their components, SREs or effector modules can be repeatedly administered without eliciting an immune response.

In another embodiment, the biocircuits, their components, SREs or effector modules is 2-fold less immunogenic than its unmodified counterpart or reference compound. In another embodiment, immunogenicity is reduced by a 3-fold factor. In another embodiment, immunogenicity is reduced by a 5-fold factor. In another embodiment, immunogenicity is reduced by a 7-fold factor. In another embodiment, immunogenicity is reduced by a 10-fold factor. In another embodiment, immunogenicity is reduced by a 15-fold factor. In another embodiment, immunogenicity is reduced by a fold factor. In another embodiment, immunogenicity is reduced by a 50-fold factor. In another embodiment, immunogenicity is reduced by a 100-fold factor. In another embodiment, immunogenicity is reduced by a 200-fold factor. In another embodiment, immunogenicity is reduced by a 500-fold factor. In another embodiment, immunogenicity is reduced by a 1000-fold factor. In another embodiment, immunogenicity is reduced by a 2000-fold factor. In another embodiment, immunogenicity is reduced by another fold difference.

Methods of determining immunogenicity are well known in the art, and include, e.g. measuring secretion of cytokines (e.g. IL12, IFNalpha, TNF-alpha, RANTES, MIP-1alpha or beta, IL6, IFN beta, or IL8), measuring expression of DC activation markers (e.g. CD83, HLA-DR, CD80 and CD86), or measuring ability to act as an adjuvant for an adaptive immune response.

The present invention provides method of inducing an immune response in a cell. As used herein the term “immune response” refers to the activity of the cells of the immune system in response to stimulus, or an antigen. In some embodiments, the antigen may be a cancer antigen. In some aspects, the methods of inducing an immune response may involve administering to the cell, a therapeutically effective amount of any of the compositions described herein. In one aspect, the method may involve administering the pharmaceutical compositions described herein. In one aspect, the method may involve administering the polynucleotides, vectors. In some embodiments, induction of the immune response occurs due to the expression and or function of the immunotherapeutic agents described herein. Methods of inducing immune response further may involve administering to the cell, an effective amount of the stimulus to tune the expression of the immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is capable of inducing an immune response in response to the stimulus. The induction of the immune response may occur in a cell within a subject i.e. in vivo, ex vivo or in vitro. The induction of an immune response may be evaluated by measuring the release of cytokine such as IL2 and IFNγ from the cells. In some embodiments, the induction of an immune response may be measured by evaluating the cell surface markers such as but not limited to CD3, CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 and HLA-DR, CD 69, CD28, CD44, IFNgamma. Examples of cell surface markers for antigen presenting cells include, but are not limited to, WIC class I, MHC Class II, CD40, CD45, B7-1, B7-2, IFN γ receptor and IL2 receptor, ICAM-1 and/or Fcγ receptor. Examples of cell surface markers for dendritic cells include, but are not limited to, WIC class I, MHC Class II, B7-2, CD18, CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR and/or Dectin-1 and the like; while in some cases also having the absence of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD 19, CD20, CD56, and/or CD57. Examples of cell surface markers for NK cells include, but are not limited to, CCL3, CCL4, CCL5, Granulysin, Granzyme B, Granzyme K, IL10, IL22, IFNg, LAP, Perforin, and TNFa.

2. Cancer Vaccines

In some embodiments, biocircuits, effector modules, payloads of interest (immunotherapeutic agents), vectors, cells and compositions of the present invention may be used for cancer vaccines. In one aspect, dendritic cells are modified to express the compositions of the invention and used as cancer vaccines.

In some embodiments, cancer vaccine may comprise peptides and/or proteins derived from tumor associated antigen (TAA). Such strategies may be utilized to evoke an immune response in a subject, which in some instances may be a cytotoxic T lymphocyte (CTL) response. Peptides used for cancer vaccines may also modified to match the mutation profile of a subject. For example, EGFR derived peptides with mutations matched to the mutations found in the subject in need of therapy have been successfully used in patients with lung cancer (Li F et al. (2016) Oncoimmunology. October 7; 5 (12): e1238539; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, cancer vaccines of the present invention may superagonist altered peptide ligands (APL) derived from TAAs. These are mutant peptide ligands deviate from the native peptide sequence by one or more amino acids, which activate specific CTL clones more effectively than native epitopes. These alterations may allow the peptide to bind better to the restricting Class I MHC molecule or interact more favorably with the TCR of a given tumor-specific CTL subset. APLs may be selected using methods taught in US Patent Publication NO.: US20160317633A1, the contents of which are incorporated herein by reference in their entirety.

3. Tumor Microenvironment (TME)

In some embodiments, biocircuits, their components, SREs or effector modules are designed to reshape the tumor microenvironment in order to extend utility of the biocircuit or a pharmaceutical composition beyond direct cell killing.

In some embodiments, biocircuits, their components, SREs or effector modules are designed to reduce, mitigate or eliminate the CAR cytokine storm. In some embodiments, such reduction, mitigation and/or elimination occurs in solid tumors or tumor microenvironments.

In some embodiments, biocircuits, effector modules, payloads of interest (immunotherapeutic agents), vectors, cells and pharmaceutical compositions of the present invention may be used to convert the immunosuppressive microenvironment to increase immune responses.

In some embodiments, the invention provides methods for converting the inhibitory immunoregulatory signals from immunosuppressive cytokines secreted by cancer cells or the surrounding tumor stroma into stimulatory signals using compositions of the invention. Immunosuppressive cytokines include without limitation, IL13, IL4, TGF-beta, IL6, IL8, and IL10. In one aspect, the genetically modified tumor-specific T cells (e.g., CAR T cells) or T cells with native tumor specificity may be further engineered to express an effector module comprising a chimeric switch receptor that binds to inhibitory/suppressive cytokines and converts their intracellular consequences to an immunostimulatory/activating signal, thus improving the efficacy of tumor-specific T cells. The chimeric switch receptor is composed of the extracellular domains of an inhibitory cytokine receptor (e.g., IL13R, IL4R, IL10R, TGFβR1/ALK5, TGFβR2, and TGFβR3/β-glycan) fused with the intracellular signal transducing domains derived from stimulatory cytokine receptors such as IL2R (i.e., IL2Rα/CD25, IL2Rβ/CD122, and common γ chain receptor/CD132 which is shared by various cytokine receptors) and/or IL7R (IL7Rα/CD127, common γ chain receptor/CD132). These manipulations render tumor specific T cells or CAR T cells resistant to the suppressive tumor microenvironment.

In some embodiments, the present invention provides methods to abrogate the immunosuppressive effects produced by myeloid derived suppressor cells (MDSCs). Tumor cells secrete indoleamine 2,3-dioxygenase (IDO) which promotes immunosuppression through the recruitment of MDSCs. The immunosuppressive environment is further promoted by the MDSCs through nitric oxide synthase (NOS) and arginase 1 (ARG1) which can degrade extracellular arginine. Amino acid deprivation within the tumor microenvironment suppresses T cell anti-tumor activity. In some embodiments, payloads of the present invention may comprise inhibitors of NOS, ARG1 and tryptophan metabolism pathway such as IDO. In one embodiment, the payload may include inhibitors of colony stimulating factor receptor 1 (CSFR1) which required for the proliferation and function of MDSCs.

In some embodiments, dominant negative mutants of inhibitory co receptor such as PD-1, CTLA-4, LAG-3, TIM-3, KIRs, or BTLA may be utilized as payloads of the invention to overcome inhibitory signals in the tumor microenvironment.

4. Combination Treatments

In some embodiments, it is desirable to combine compositions, vectors and cells of the invention for administration to a subject. Compositions of the invention comprising different immunotherapeutic agents may be used in combination for enhancement of immunotherapy.

Immunotherapeutic Agents

In some embodiments, it is desirable to combine compositions of the invention with adjuvants, that can enhance the potency and longevity of antigen-specific immune responses. Adjuvants used as immunostimulants in combination therapy include biological molecules or delivery carriers that deliver antigens. As non-limiting examples, the compositions of the invention may be combined with biological adjuvants such as cytokines, Toll Like Receptors, bacterial toxins, and/or saponins. In other embodiments, the compositions of the present invention may be combined with delivery carriers. Exemplary delivery carriers include, polymer microspheres, immune stimulating complexes, emulsions (oil-in-water or water-in-oil), aluminum salts, liposomes or virosomes.

In some embodiments, immune effector cells modified to express biocircuits, effector modules, DDs and payloads of the invention may be combined with the biological adjuvants described herein. Dual regulation of CAR and cytokines and ligands to segregate the kinetic control of target-mediated activation from intrinsic cell T cell expansion. Such dual regulation also minimizes the need for pre-conditioning regimens in patients. As a non-limiting example, DD regulated CAR e.g. CD19 CAR may be combined with cytokines e.g. IL12 to enhance the anti-tumor efficacy of the CAR (Pegram H. J., et al. Tumor-targeted T cells modified to secrete IL12 eradicate systemic tumors without need for prior conditioning. Blood. 2012; 119:4133-41; the contents of each of which are incorporated herein by reference in their entirety). As another non-limiting example, Merchant et al. combined dendritic cell-based vaccinations with recombinant human IL7 to improve outcome in high-risk pediatric sarcomas patients (Merchant, M. S. et. al. Adjuvant immunotherapy to Improve Outcome in High-Risk Pediatric Sarcomas. Clin Cancer Res. 2016. 22(13):3182-91; the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, immune effector cells modified to express one or more antigen-specific TCRs or CARs may be combined with compositions of the invention comprising immunotherapeutic agents that convert the immunosuppressive tumor microenvironment.

In one aspect, effector immune cells modified to express CARs specific to different target molecules on the same cell may be combined. In another aspect, different immune cells modified to express the same CAR construct such as NK cells and T cells may be used in combination for a tumor treatment, for instance, a T cell modified to express a CD19 CAR may be combined with a NK cell modified to express the same CD19 CAR to treat B cell malignancy.

In other embodiments, immune cells modified to express CARs may be combined with checkpoint blockade agents.

In some embodiments, immune effector cells modified to expressed biocircuits, effector modules, DDs and payloads of the invention may be combined with cancer vaccines of the invention.

In some embodiments, an effector module comprising a CAR may be used in combination with an effector module comprising a cytokine, or an effector module comprising a safety switch, or an effector module comprising a metabolic factor, or an effector module comprising a homing receptor.

In one embodiment, an effector module comprising a CD19 CAR may be used in combination with amino pyrimidine derivatives such as the Burkit's tyrosine receptor kinase (BTK) inhibitor using methods taught in International Patent Application NO.: WO2016164580, the contents of which are incorporated herein by reference in their entirety.

Cancer

In some embodiments, methods of the invention may include combination of the compositions of the invention with other agents effective in the treatment of cancers, infection diseases and other immunodeficient disorders, such as anti-cancer agents. As used herein, the term “anti-cancer agent” refers to any agent which is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

In some embodiments, anti-cancer agent or therapy may be a chemotherapeutic agent, or radiotherapy, immunotherapeutic agent, surgery, or any other therapeutic agent which, in combination with the present invention, improves the therapeutic efficacy of treatment.

In some embodiments, compositions of the present invention may be used in combination with immunotherapeutics other than the inventive therapy described herein, such as antibodies specific to some target molecules on the surface of a tumor cell.

Exemplary chemotherapies include, without limitation, Acivicin; Aclarubicin; Acodazole hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone acetate; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperrin, Sulindac, Curcumin, alkylating agents including: Nitrogen mustards such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas such as carmustine (BC U), lomustine (CCNU), and semustine (methyl-CC U); thylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrrolidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and teniposide; antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta and GM-CSF, anti-angiogenic factors, such as angiostatin and endostatin, inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors, including soluble VGF/VEGF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIFf) and procarbazine, adrenocortical suppressants such as mitotane (o,ρ′-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab; Sdi 1 mimetics; Semustine; Senescence derived inhibitor 1; Sparfosic acid; Spicamycin D; Spiromustine; Splenopentin; Spongistatin 1; Squalamine; Stipiamide; Stromelysin inhibitors; Sulfinosine; Superactive vasoactive intestinal peptide antagonist; Velaresol; Veramine; Verdins; Verteporfin; Vinorelbine; Vinxaltine; Vitaxin; Vorozole; Zanoterone; Zeniplatin; Zilascorb; and Zinostatin stimalamer; PI3Kβ small-molecule inhibitor, GSK2636771; pan-PI3K inhibitor (BKM120); BRAF inhibitors. Vemurafenib (Zelboraf) and dabrafenib (Tafinlar); or any analog or derivative and variant of the foregoing.

In one embodiment, the invention further relates to the use of pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention for treating one or more forms of cancer, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, the pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention can also be administered in conjunction with one or more additional anti-cancer treatments, such as biological, chemotherapy and radiotherapy. Accordingly, a treatment can include, for example, imatinib (Gleevac), all-trans-retinoic acid, a monoclonal antibody treatment (gemtuzumab, ozogamicin), chemotherapy (for example, chlorambucil, prednisone, prednisolone, vincristine, cytarabine, clofarabine, farnesyl transferase inhibitors, decitabine, inhibitors of MDR1), rituximab, interferon-α, anthracycline drugs (such as daunorubicin or idarubicin), L-asparaginase, doxorubicin, cyclophosphamide, doxorubicin, bleomycin, fludarabine, etoposide, pentostatin, or cladribine), bone marrow transplant, stem cell transplant, radiation therapy, anti-metabolite drugs (methotrexate and 6-mercaptopurine), or any of the antibodies taught herein such as those in Table 6 of the co-owned U.S. Provisional Patent Application No. 62/320,864 filed Apr. 11, 2016, 62/466,596 filed Mar. 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety), or combinations thereof.

Radiotherapeutic agents and factors include radiation and waves that induce DNA damage for example, γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

Radiation therapy (also called radiotherapy, X-ray therapy, or irradiation) is the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered externally via external beam radiotherapy (EBRT) or internally via brachytherapy. The effects of radiation therapy are localized and confined to the region being treated. Radiation therapy may be used to treat almost every type of solid tumor, including cancers of the brain, breast, cervix, larynx, lung, pancreas, prostate, skin, stomach, uterus, or soft tissue sarcomas. Radiation is also used to treat leukemia and lymphoma.

In some embodiments, the chemotherapeutic agent may be an immunomodulatory agent such as lenalidomide (LEN). Recent studies have demonstrated that lenalidomide can enhance antitumor functions of CAR modified T cells (Otahal et al., Oncoimmunology, 2015, 5(4): e1115940). Some examples of anti-tumor antibodies include tocilizumab, siltuximab.

Chemotherapy is the treatment of cancer with drugs that can destroy cancer cells. In current usage, the term “chemotherapy” usually refers to cytotoxic drugs which affect rapidly dividing cells in general, in contrast with targeted therapy. Chemotherapy drugs interfere with cell division in various possible ways, e.g. with the duplication of DNA or the separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly dividing cells and are not specific to cancer cells, although some degree of specificity may come from the inability of many cancer cells to repair DNA damage, while normal cells generally can.

Most chemotherapy regimens are given in combination. Exemplary chemotherapeutic agents include, but are not limited to, 5-FU Enhancer, 9-AC, AG2037, AG3340, Aggrecanase Inhibitor, Aminoglutethimide, Amsacrine (m-AMSA), Asparaginase, Azacitidine, Batimastat (BB94), BAY 12-9566, BCH-4556, Bis-Naphthalimide, Busulfan, Capecitabine, Carboplatin, Carmustaine+Polifepr Osan, cdk4/cdk2 inhibitors, Chlorambucil, CI-994, Cisplatin, Cladribine, CS-682, Cytarabine HCl, D2163, Dactinomycin, Daunorubicin HCl, DepoCyt, Dexifosamide, Docetaxel, Dolastain, Doxifluridine, Doxorubicin, DX8951f, E 7070, EGFR, Epirubicin, Erythropoietin, Estramustine phosphate sodium, Etoposide (VP16-213), Farnesyl Transferase Inhibitor, FK 317, Flavopiridol, Floxuridine, Fludarabine, Fluorouracil (5-FU), Flutamide, Fragyline, Gemcitabine, Hexamethylmelamine (HMM), Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Interleukin-2, Irinotecan, ISI 641, Krestin, Lemonal DP 2202, Leuprolide acetate (LHRH-releasing factor analogue), Levamisole, LiGLA (lithium-gamma linolenate), Lodine Seeds, Lometexol, Lomustine (CCNU), Marimistat, Mechlorethamine HCl (nitrogen mustard), Megestrol acetate, Meglamine GLA, Mercaptopurine, Mesna, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Mitotane (o.ρ′-DDD), Mitoxantrone, Mitoxantrone HCl, MMI 270, MMP, MTA/LY 231514, Octreotide, ODN 698, OK-432, Oral Platinum, Oral Taxoid, Paclitaxel (TAXOL.RTM.), PARP Inhibitors, PD 183805, Pentostatin (2′ deoxycoformycin), PKC 412, Plicamycin, Procarbazine HCl, PSC 833, Ralitrexed, RAS Farnesyl Transferase Inhibitor, RAS Oncogene Inhibitor, Semustine (methyl-CCNU), Streptozocin, Suramin, Tamoxifen citrate, Taxane Analog, Temozolomide, Teniposide (VM-26), Thioguanine, Thiotepa, Topotecan, Tyrosine Kinase, UFT (Tegafur/Uracil), Valrubicin, Vinblastine sulfate, Vindesine sulfate, VX-710, VX-853, YM 116, ZD 0101, ZD 0473/Anormed, ZD 1839, ZD 9331.

Other agents may be used in combination with compositions of the invention may also include, but not limited to, agents that affect the upregulation of cell surface receptors and their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion such as focal adhesion kinase (FAKs) inhibitors and Lovastatin, or agents that increase the sensitivity of the hyper proliferative cells to apoptotic inducers such as the antibody C225.

The combinations may include administering the compositions of the invention and other agents at the same time or separately. Alternatively, the present immunotherapy may precede or follow the other agent/therapy by intervals ranging from minutes, days, weeks to months.

5. Therapeutic Uses

Provided in the present invention is a method of reducing a tumor volume or burden in a subject in need, the method comprising introducing into the subject a composition of the invention.

The present invention also provides methods for treating a cancer in a subject, comprising administering to the subject an effective amount of an immune effector cell genetically modified to express at least one effector module of the invention.

Cancer

Various cancers may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As used herein, the term “cancer” refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of lymphomas/leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.

Types of carcinomas which may be treated with the compositions of the present invention include, but are not limited to, papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.

Types of carcinomas which may be treated with the compositions of the present invention include, but are not limited to, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

As a non-limiting example, the carcinoma which may be treated may be Acute granulocytic leukemia, Acute lymphocytic leukemia, Acute myelogenous leukemia, Adenocarcinoma, Adenosarcoma, Adrenal cancer, Adrenocortical carcinoma, Anal cancer, Anaplastic astrocytoma, Angiosarcoma, Appendix cancer, Astrocytoma, Basal cell carcinoma, B-Cell lymphoma), Bile duct cancer, Bladder cancer, Bone cancer, Bowel cancer, Brain cancer, Brain stem glioma, Brain tumor, Breast cancer, Carcinoid tumors, Cervical cancer, Cholangiocarcinoma, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Colon cancer, Colorectal cancer, Craniopharyngioma, Cutaneous lymphoma, Cutaneous melanoma, Diffuse astrocytoma, Ductal carcinoma in situ, Endometrial cancer, Ependymoma, Epithelioid sarcoma, Esophageal cancer, Ewing sarcoma, Extrahepatic bile duct cancer, Eye cancer, Fallopian tube cancer, Fibrosarcoma, Gallbladder cancer, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid cancer, Gastrointestinal stromal tumors, General, Germ cell tumor, Glioblastoma multiforme, Glioma, Hairy cell leukemia, Head and neck cancer, Hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, Hodgkin's lymphoma, Hypopharyngeal cancer, Infiltrating ductal carcinoma, Infiltrating lobular carcinoma, Inflammatory breast cancer, Intestinal Cancer, Intrahepatic bile duct cancer, Invasive/infiltrating breast cancer, Islet cell cancer, Jaw cancer, Kaposi sarcoma, Kidney cancer, Laryngeal cancer, Leiomyosarcoma, Leptomeningeal metastases, Leukemia, Lip cancer, Liposarcoma, Liver cancer, Lobular carcinoma in situ, Low-grade astrocytoma, Lung cancer, Lymph node cancer, Lymphoma, Male breast cancer, Medullary carcinoma, Medulloblastoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesenchymal chondrosarcoma, Mesenchymous, Mesothelioma, Metastatic breast cancer, Metastatic melanoma, Metastatic squamous neck cancer, Mixed gliomas, Mouth cancer, Mucinous carcinoma, Mucosal melanoma, Multiple myeloma, Nasal cavity cancer, Nasopharyngeal cancer, Neck cancer, Neuroblastoma, Neuroendocrine tumors, Non-Hodgkin lymphoma, Non-Hodgkin's lymphoma, Non-small cell lung cancer, Oat cell cancer, Ocular cancer, Ocular melanoma, Oligodendroglioma, Oral cancer, Oral cavity cancer, Oropharyngeal cancer, Osteogenic sarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Ovarian primary peritoneal carcinoma, Ovarian sex cord stromal tumor, Paget's disease, Pancreatic cancer, Papillary carcinoma, Paranasal sinus cancer, Parathyroid cancer, Pelvic cancer, Penile cancer, Peripheral nerve cancer, Peritoneal cancer, Pharyngeal cancer, Pheochromocytoma, Pilocytic astrocytoma, Pineal region tumor, Pineoblastoma, Pituitary gland cancer, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell cancer, Renal pelvis cancer, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sarcoma, bone, Sarcoma, soft tissue, Sarcoma, uterine, Sinus cancer, Skin cancer, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Spinal cancer, Spinal column cancer, Spinal cord cancer, Spinal tumor, Squamous cell carcinoma, Stomach cancer, Synovial sarcoma, T-cell lymphoma), Testicular cancer, Throat cancer, Thymoma/thymic carcinoma, Thyroid cancer, Tongue cancer, Tonsil cancer, Transitional cell cancer, Transitional cell cancer, Transitional cell cancer, Triple-negative breast cancer, Tubal cancer, Tubular carcinoma, Ureteral cancer, Ureteral cancer, Urethral cancer, Uterine adenocarcinoma, Uterine cancer, Uterine sarcoma, Vaginal cancer, and Vulvar cancer.

In some embodiments, the CARs of the present invention may be a CAR useful in the treatment of multiple myeloma such as a CS1 CAR, a CD38 CAR, a CD138 CAR, and a BCMA CAR. In some embodiments, the CARs of the present invention may be a CAR useful in the treatment of acute myeloid leukemia such as a CD33 CAR, a CD123 CAR, and a CLL1 CAR. In some embodiments, the CARs of the present invention may be a CAR useful in the treatment of T cell leukemia such as a CD5 CAR, and a CD7 CAR. In some embodiments, the CARs of the present invention may be a CAR useful in the treatment of solid tumors such a mesothelin CAR, a GD2 CAR, a GPC3 CAR, a Her2 CAR, an EGFR CAR, a Mud CAR, an EpCAM CAR, a PD-L1 CAR, a CEA CAR, a Muc16 CAR, a CD133 CAR, a CD171 CAR, a CD70 CAR, a CLD18 CAR, a cMET CAR, a EphA2 CAR, a FAP CAR, a Folate Receptor CAR, an IL13Rα2 CAR, an MG7 CAR, a PSMA CAR, a ROR1 CAR, and a VEGFR2 CAR.

The present invention also provides methods of reducing tumor burden in a subject. In some embodiments. As used herein, “tumor burden” refers to the number of cancer cells, or the amount of cancer in a subject. In some aspects tumor burden also refers to tumor load. In some embodiments, the tumor may be disseminated throughout the body of the subject. In one aspect, the tumor may be a liquid tumor such as leukemia or a lymphoma. The methods of reducing tumor burden may involve administering to the subject, a therapeutically effective amount of the immune cells. Immune cells may be engineered to express the compositions described herein. In some embodiments, the immune cells expressing the compositions of the invention may be administered to the subject via any of the routes of delivery described herein. Also provided herein are dosing regimens for administering the immune cells. In some embodiments, the subject may also be administered a therapeutically effective amount of the stimulus to tune the expression of the immunotherapeutic agent. In some aspects, the immunotherapeutic agents may be capable of reducing the tumor burden. Regimens for ligand/stimulus dosing are also provided. Reduction in tumor burden may be measured by any of the methods known in the art including tumor imaging, and measurement of marker proteins. In some aspects, bioluminescent imaging may be used to measure tumor burden. Bioluminescence imaging utilizes native light emission from bioluminescent proteins such as luciferase. Such bioluminescent proteins can participate in chemical reactions that release photons by the addition of suitable substrates. The release of photons can be captured by sensitive detection methods and quantified. Tumor cells may be engineered to express luciferase and the efficacy of the compositions described herein to reduce tumor burden may quantified by imaging. In some aspects, the tumor burden may be measured by the flux of photons (photons per sec). In some embodiments, photon flux positively correlates with tumor burden.

Diseases and Toxins

Various infectious diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As used herein, the term “infectious disease” refers to any disorders caused by organisms such as bacteria, viruses, fungi or parasites. As a non-limiting example, the infectious disease may be Acute bacterial rhinosinusitis, 14-day measles, Acne, Acrodermatitis chronica atrophicans (ACA)-(late skin manifestation of latent Lyme disease), Acute hemorrhagic conjunctivitis, Acute hemorrhagic cystitis, Acute rhinosinusitis, Adult T-cell Leukemia-Lymphoma (ATLL), African Sleeping Sickness, AIDS (Acquired Immunodeficiency Syndrome), Alveolar hydatid, Amebiasis, Amebic meningoencephalitis, Anaplasmosis, Anthrax, Arboviral or parainfectious, Ascariasis-(Roundworm infections), Aseptic meningitis, Athlete's foot (Tinea pedis), Australian tick typhus, Avian Influenza, Babesiosis, Bacillary angiomatosis, Bacterial meningitis, Bacterial vaginosis, Balanitis, Balantidiasis, Bang's disease, Barmah Forest virus infection, Bartonellosis (Verruga peruana; Carrion's disease; Oroya fever), Bat Lyssavirus Infection, Bay sore (Chiclero's ulcer), Baylisascaris infection (Racoon roundworm infection), Beaver fever, Beef tapeworm, Bejel (endemic syphilis), Biphasic meningoencephalitis, Black Bane, Black death, Black piedra, Blackwater Fever, Blastomycosis, Blennorrhea of the newborn, Blepharitis, Boils, Bornholm disease (pleurodynia), Borrelia miyamotoi Disease, Botulism, Boutonneuse fever, Brazilian purpuric fever, Break Bone fever, Brill, Bronchiolitis, Bronchitis, Brucellosis (Bang's disease), Bubonic plague, Bullous impetigo, Burkholderia mallei (Glanders), Burkholderia pseudomallei (Melioidosis), Buruli ulcers (also Mycoburuli ulcers), Busse, Busse-Buschke disease (Cryptococcosis), California group encephalitis, Campylobacteriosis, Candidiasis, Canefield fever (Canicola fever; 7-day fever; Weil's disease; leptospirosis; canefield fever), Canicola fever, Capillariasis, Carate, Carbapenem-resistant Enterobacteriaceae (CRE), Carbuncle, Carrion's disease, Cat Scratch fever, Cave disease, Central Asian hemorrhagic fever, Central European tick, Cervical cancer, Chagas disease, Chancroid (Soft chancre), Chicago disease, Chickenpox (Varicella), Chiclero's ulcer, Chikungunya fever, Chlamydial infection, Cholera, Chromoblastomycosis, Ciguatera, Clap, Clonorchiasis (Liver fluke infection), Clostridium Difficile Infection, Clostridium Perfringens (Epsilon Toxin), Coccidioidomycosis fungal infection (Valley fever; desert rheumatism), Coenurosis, Colorado tick fever, Condyloma accuminata, Condyloma accuminata (Warts), Condyloma lata, Congo fever, Congo hemorrhagic fever virus, Conjunctivitis, cowpox, Crabs, Crimean, Croup, Cryptococcosis, Cryptosporidiosis (Crypto), Cutaneous Larval Migrans, Cyclosporiasis, Cystic hydatid, Cysticercosis, Cystitis, Czechoslovak tick, D68 (EV-D68), Dacryocytitis, Dandy fever, Darling's Disease, Deer fly fever, Dengue fever (1, 2, 3 and 4), Desert rheumatism, Devil's grip, Diphasic milk fever, Diphtheria, Disseminated Intravascular Coagulation, Dog tapeworm, Donovanosis, Donovanosis (Granuloma inguinale), Dracontiasis, Dracunculosis, Duke's disease, Dum Dum Disease, Durand-Nicholas-Favre disease, Dwarf tapeworm, E. Coli infection (E. coli), Eastern equine encephalitis, Ebola Hemorrhagic Fever (Ebola virus disease EVD), Ectothrix, Ehrlichiosis (Sennetsu fever), Encephalitis, Endemic Relapsing fever, Endemic syphilis, Endophthalmitis, Endothrix, Enterobiasis (Pinworm infection), Enterotoxin—B Poisoning (Staph Food Poisoning), Enterovirus Infection, Epidemic Keratoconjunctivitis, Epidemic Relapsing fever, Epidemic typhus, Epiglottitis, Erysipelis, Erysipeloid (Erysipelothricosis), Erythema chronicum migrans, Erythema infectiosum, Erythema marginatum, Erythema multiforme, Erythema nodosum, Erythema nodosum leprosum, Erythrasma, Espundia, Eumycotic mycetoma, European blastomycosis, Exanthem subitum (Sixth disease), Eyeworm, Far Eastern tick, Fascioliasis, Fievre boutonneuse (Tick typhus), Fifth Disease (erythema infectiosum), Filatow-Dukes' Disease (Scalded Skin Syndrome; Ritter's Disease), Fish tapeworm, Fitz-Hugh-Curtis syndrome—Perihepatitis, Flinders Island Spotted Fever, Flu (Influenza), Folliculitis, Four Corners Disease, Four Corners Disease (Human Pulmonary Syndrome (HPS)), Frambesia, Francis disease, Furunculosis, Gas gangrene, Gastroenteritis, Genital Herpes, Genital Warts, German measles, Gerstmann-Straussler-Scheinker (GSS), Giardiasis, Gilchrist's disease, Gingivitis, Gingivostomatitis, Glanders, Glandular fever (infectious mononucleosis), Gnathostomiasis, Gonococcal Infection (Gonorrhea), Gonorrhea, Granuloma inguinale (Donovanosis), Guinea Worm, Haemophilus Influenza disease, Hamburger disease, Hansen's disease—leprosy, Hantaan disease, Hantaan-Korean hemorrhagic fever, Hantavirus Pulmonary Syndrome, Hantavirus Pulmonary Syndrome (HPS), Hard chancre, Hard measles, Haverhill fever—Rat bite fever, Head and Body Lice, Heartland fever, Helicobacterosis, Hemolytic Uremic Syndrome (HUS), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpangina, Herpes—genital, Herpes labialis, Herpes—neonatal, Hidradenitis, Histoplasmosis, Histoplasmosis infection (Histoplasmosis), His-Werner disease, HIV infection, Hookworm infections, Hordeola, Hordeola (Stye), HTLV, HTLV associated myelopathy (HAM), Human granulocytic ehrlichiosis, Human monocytic ehrlichiosis, Human Papillomavirus (HPV), Human Pulmonary Syndrome, Hydatid cyst, Hydrophobia, Impetigo, Including congenital (German Measles), Inclusion conjunctivitis, Inclusion conjunctivitis—Swimming Pool conjunctivitis-Pannus, Infantile diarrhea, Infectious Mononucleosis, Infectious myocarditis, Infectious pericarditis, Influenza, Isosporiasis, Israeli spotted fever, Japanese Encephalitis, Jock itch, Jorge Lobo disease—lobomycosis, Jungle yellow fever, Junin Argentinian hemorrhagic fever, Kala Azar, Kaposi's sarcoma, Keloidal blastomycosis, Keratoconjunctivitis, Kuru, Kyasanur forest disease, LaCrosse encephalitis, Lassa hemorrhagic fever, Legionellosis (Legionnaires Disease), Legionnaire's pneumonia, Lemierre's Syndrome (Postanginal septicemia), Lemming fever, Leprosy, Leptospirosis (Nanukayami fever; Weil's disease), Listeriosis (Listeria), Liver fluke infection, Lobo's mycosis, Lockjaw, Loiasis, Louping Ill, Ludwig's angina, Lung fluke infection, Lung fluke infection (Paragonimiasis), Lyme disease, Lymphogranuloma venereum infection (LGV), Machupo Bolivian hemorrhagic fever, Madura foot, Mal del pinto, Malaria, Malignant pustule, Malta fever, Marburg hemorrhagic fever, Masters disease, Maternal Sepsis (Puerperal fever), Measles, Mediterranean spotted fever, Melioidosis (Whitmore's disease), Meningitis, Meningococcal Disease, MERS, Milker's nodule, Molluscum contagiosum, Moniliasis, monkeypox, Mononucleosis, Mononucleosis-like syndrome, Montezuma's Revenge, Morbilli, MRSA (methicillin-resistant Staphylococcus aureus) infection, Mucormycosis-Zygomycosis, Multiple Organ Dysfunction Syndrome or MODS, Multiple-system atrophy (MSA), Mumps, Murine typhus, Murray Valley Encephalitis (MVE), Mycoburuli ulcers, Mycoburuli ulcers-Buruli ulcers, Mycotic vulvovaginitis, Myositis, Nanukayami fever, Necrotizing fasciitis, Necrotizing fasciitis-Type 1, Necrotizing fasciitis-Type 2, Negishi, New world spotted fever, Nocardiosis, Nongonococcal urethritis, Non-Polio (Non-Polio Enterovirus), Norovirus infection, North American blastomycosis, North Asian tick typhus, Norwalk virus infection, Norwegian itch, O'Hara disease, Omsk hemorrhagic fever, Onchoceriasis, Onychomycosis, Opisthorchiasis, Opthalmia neonatorium, Oral hairy leukoplakia, Orf, Oriental Sore, Oriental Spotted Fever, Ornithosis (Parrot fever; Psittacosis), Oroya fever, Otitis externa, Otitis media, Pannus, Paracoccidioidomycosis, Paragonimiasis, Paralytic Shellfish Poisoning (Paralytic Shellfish Poisoning), Paronychia (Whitlow), Parotitis, PCP pneumonia, Pediculosis, Peliosis hepatica, Pelvic Inflammatory Disease, Pertussis (also called Whooping cough), Phaeohyphomycosis, Pharyngoconjunctival fever, Piedra (White Piedra), Piedra (Black Piedra), Pigbel, Pink eye conjunctivitis, Pinta, Pinworm infection, Pitted Keratolysis, Pityriasis versicolor (Tinea versicolor), Plague; Bubonic, Pleurodynia, Pneumococcal Disease, Pneumocystosis, Pneumonia, Pneumonic (Plague), Polio or Poliomyelitis, Polycystic hydatid, Pontiac fever, Pork tapeworm, Posada-Wernicke disease, Postanginal septicemia, Powassan, Progressive multifocal leukoencephalopathy, Progressive Rubella Panencephalitis, Prostatitis, Pseudomembranous colitis, Psittacosis, Puerperal fever, Pustular Rash diseases (Small pox), Pyelonephritis, Pylephlebitis, Q-Fever, Quinsy, Quintana fever (5-day fever), Rabbit fever, Rabies, Racoon roundworm infection, Rat bite fever, Rat tapeworm, Reiter Syndrome, Relapsing fever, Respiratory syncytial virus (RSV) infection, Rheumatic fever, Rhodotorulosis, Ricin Poisoning, Rickettsialpox, Rickettsiosis, Rift Valley Fever, Ringworm, Ritter's Disease, River Blindness, Rocky Mountain spotted fever, Rose Handler's disease (Sporotrichosis), Rose rash of infants, Roseola, Ross River fever, Rotavirus infection, Roundworm infections, Rubella, Rubeola, Russian spring, Salmonellosis gastroenteritis, San Joaquin Valley fever, Sao Paulo Encephalitis, Sao Paulo fever, SARS, Scabies Infestation (Scabies) (Norwegian itch), Scalded Skin Syndrome, Scarlet fever (Scarlatina), Schistosomiasis, Scombroid, Scrub typhus, Sennetsu fever, Sepsis (Septic shock), Severe Acute Respiratory Syndrome, Severe Acute Respiratory Syndrome (SARS), Shiga Toxigenic Escherichia coli (STEC/VTEC), Shigellosis gastroenteritis (Shigella), Shinbone fever, Shingles, Shipping fever, Siberian tick typhus, Sinusitis, Sixth disease, Slapped cheek disease, Sleeping sickness, Smallpox (Variola), Snail Fever, Soft chancre, Southern tick associated rash illness, Sparganosis, Spelunker's disease, Sporadic typhus, Sporotrichosis, Spotted fever, Spring, St. Louis encephalitis, Staphylococcal Food Poisoning, Staphylococcal Infection, Strep. throat, Streptococcal Disease, Streptococcal Toxic-Shock Syndrome, Strongyloiciasis, Stye, Subacute Sclerosing Panencephalitis, Subacute Sclerosing Panencephalitis (SSPE), Sudden Acute Respiratory Syndrome, Sudden Rash, Swimmer's ear, Swimmer's Itch, Swimming Pool conjunctivitis, Sylvatic yellow fever, Syphilis, Systemic Inflammatory Response Syndrome (SIRS), Tabes dorsalis (tertiary syphilis), Taeniasis, Taiga encephalitis, Tanner's disease, Tapeworm infections, Temporal lobe encephalitis, Temporal lobe encephalitis, tetani (Lock Jaw), Tetanus Infection, Threadworm infections, Thrush, Tick, Tick typhus, Tinea barbae, Tinea capitis, Tinea corporis, Tinea cruris, Tinea manuum, Tinea nigra, Tinea pedis, Tinea unguium, Tinea versicolor, Torulopsosis, Torulosis, Toxic Shock Syndrome, Toxoplasmosis, transmissible spongioform (CJD), Traveler's diarrhea, Trench fever 5, Trichinellosis, Trichomoniasis, Trichomycosis axillaris, Trichuriasis, Tropical Spastic Paraparesis (TSP), Trypanosomiasis, Tuberculosis (TB), Tuberculosis, Tularemia, Typhoid Fever, Typhus fever, Ulcus molle, Undulant fever, Urban yellow fever, Urethritis, Vaginitis, Vaginosis, Vancomycin Intermediate (VISA), Vancomycin Resistant (VRSA), Varicella, Venezuelan Equine encephalitis, Verruga peruana, Vibrio cholerae (Cholera), Vibriosis (Vibrio), Vincent's disease or Trench mouth, Viral conjunctivitis, Viral Meningitis, Viral meningoencephalitis, Viral rash, Visceral Larval Migrans, Vomito negro, Vulvovaginitis, Warts, Waterhouse, Weil's disease, West Nile Fever, Western equine encephalitis, Whipple's disease, Whipworm infection, White Piedra, Whitlow, Whitmore's disease, Winter diarrhea, Wolhynia fever, Wool sorters' disease, Yaws, Yellow Fever, Yersinosis, Yersinosis (Yersinia), Zahorsky's disease, Zika virus disease, Zoster, Zygomycosis, John Cunningham Virus (JCV), Human immunodeficiency virus (HIV), Influenza virus, Hepatitis B, Hepatitis C, Hepatitis D, Respiratory syncytial virus (RSV), Herpes simplex virus 1 and 2, Human Cytomegalovirus, Epstein-Barr virus, Varicella zoster virus, Coronaviruses, Poxviruses, Enterovirus 71, Rubella virus, Human papilloma virus, Streptococcus pneumoniae, Streptococcus viridans, Staphylococcus aureus (S. aureus), Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-intermediate Staphylococcus aureus (VISA), Vancomycin-resistant Staphylococcus aureus (VRSA), Staphylococcus epidermidis (S. epidermidis), Clostridium Tetani, Bordetella pertussis, Bordetella paratussis, Mycobacterium, Francisella tularensis, Toxoplasma gondii, Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei and C. lusitaniae) and/or any other infectious diseases, disorders or syndromes.

Various toxins may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. Non-limited examples of toxins include Ricin, Bacillus anthracis, Shiga toxin and Shiga-like toxin, Botulinum toxins.

Various tropical diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. Non-limited examples of tropical diseases include Chikungunya fever, Dengue fever, Chagas disease, Rabies, Malaria, Ebola virus, Marburg virus, West Nile Virus, Yellow Fever, Japanese encephalitis virus, St. Louis encephalitis virus.

Various foodborne illnesses and gastroenteritis may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. Non-limited examples of foodborne illnesses and gastroenteritis include Rotavirus, Norwalk virus (Norovirus), Campylobacter jejuni, Clostridium difficile, Entamoeba histolytica, Helicobacter pylori, Enterotoxin B of Staphylococcus aureus, Hepatitis A virus (HAV), Hepatitis E, Listeria monocytogenes, Salmonella, Clostridium perfringens, and Salmonella.

Various infectious agents may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. Non-limited examples of infectious agents include adenoviruses, Anaplasma phagocytophilium, Ascaris lumbricoides, Bacillus anthracis, Bacillus cereus, Bacteroides sp, Barmah Forest virus, Bartonella bacilliformis, Bartonella henselae, Bartonella quintana, beta-toxin of Clostridium perfringens, Bordetella pertussis, Bordetella parapertussis, Borrelia burgdorferi, Borrelia miyamotoi, Borrelia recurrentis, Borrelia sp., Botulinum toxin, Brucella sp., Burkholderia pseudomallei, California encephalitis virus, Campylobacter, Candida albicans, chikungunya virus, Chlamydia psittaci, Chlamydia trachomatis, Clonorchis sinensis, Clostridium difficile bacteria, Clostridium tetani, Colorado tick fever virus, Corynebacterium diphtherias, Corynebacterium minutissimum, Coxiella burnetii, coxsackie A, coxsackie B, Crimean-Congo hemorrhagic fever virus, cytomegalovirus, dengue virus, Eastern Equine encephalitis virus, Ebola viruses, echovirus, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia sp., Entamoeba histolytica, Enterobacter sp., Enterococcus faecalis, Enterovirus 71, Epstein-Barr virus (EBV), Erysipelothrix rhusiopathiae, Escherichia coli, Flavivirus, Fusobacterium necrophorum, Gardnerella vaginalis, Group B streptococcus, Haemophilus aegyptius, Haemophilus ducreyi, Haemophilus influenzae, hantavirus, Helicobacter pylori, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, herpes simplex virus 1 and 2, human herpes virus 6, human herpes Virus 8, human immunodeficiency virus 1 and 2, human T-cell leukemia viruses I and II, influenza viruses (A, B, C), Jamestown Canyon virus, Japanese encephalitis antigenic, Japanese encephalitis virus, John Cunninham virus, juninvirus, Kaposi's Sarcoma-associated Herpes Virus (KSHV), Klebsiella granulomatis, Klebsiella sp Kyasanur Forest Disease virus, La Crosse virus, Lassavirus, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, lymphocytic choriomeningitis virus, lyssavirus, Machupovirus, Marburg virus, measles virus, MERS coronavirus (MERS-CoV), Micrococcus sedentarius, Mobiluncus sp., Molluscipoxvirus, Moraxella catarrhalis, Morbilli-Rubeola virus, Mumps virus, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma genitalium, Mycoplasma sp, Nairovirus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia, Norwalk virus, norovirus, Omsk hemorrhagic fever virus, papilloma virus, parainfluenza viruses 1-3, parapoxvirus, parvovirus B19, Peptostreptococccus sp., Plasmodium sp., polioviruses types I, II, and III, Proteus sp., Pseudomonas aeruginosa, Pseudomonas pseudomallei, Pseudomonas sp., rabies virus, respiratory syncytial virus, ricin toxin, Rickettsia australis, Rickettsia conori, Rickettsia honei, Rickettsia prowazekii, Ross River Virus, rotavirus, rubellavirus, Saint Louis encephalitis, Salmonella typhi, Sarcoptes scabiei, SARS-associated coronavirus (SARS-CoV), Serratia sp., Shiga toxin and Shiga-like toxin, Shigella sp., Sin Nombre Virus, Snowshoe hare virus, Staphylococcus aureus, Staphylococcus epidermidis, Streptobacillus moniliformis, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus agalactiae, Streptococcus group A-H, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum subsp. Pallidum, Treponema pallidum var. carateum, Treponema pallidum var. endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Varicella-Zoster virus, variola virus, Vibrio cholerae, West Nile virus, yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Zika virus.

Various rare diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As used herein, the term “rare disease” refers to any disease that affects a small percentage of the population. As a non-limiting example, the rare disease may be Acrocephalosyndactylia, Acrodermatitis, Addison Disease, Adie Syndrome, Alagille Syndrome, Amylose, Amyotrophic Lateral Sclerosis, Angelman Syndrome, Angiolymphoid Hyperplasia with Eosinophilia, Arnold-Chiari Malformation, Arthritis, Juvenile Rheumatoid, Asperger Syndrome, Bardet-Biedl Syndrome, Barrett Esophagus, Beckwith-Wiedemann Syndrome, Behcet Syndrome, Bloom Syndrome, Bowen's Disease, Brachial Plexus Neuropathies, Brown-Sequard Syndrome, Budd-Chiari Syndrome, Burkitt Lymphoma, Carcinoma 256, Walker, Caroli Disease, Charcot-Marie-Tooth Disease, Chediak-Higashi Syndrome, Chiari-Frommel Syndrome, Chondrodysplasia Punctata, Colonic Pseudo-Obstruction, Colorectal Neoplasms, Hereditary Nonpolyposis, Craniofacial Dysostosis, Creutzfeldt-Jakob Syndrome, Crohn Disease, Cushing Syndrome, Cystic Fibrosis, Dandy-Walker Syndrome, De Lange Syndrome, Dementia, Vascular, Dermatitis Herpetiformis, DiGeorge Syndrome, Diffuse Cerebral Sclerosis of Schilder, Duane Retraction Syndrome, Dupuytren Contracture, Ebstein Anomaly, Eisenmenger Complex, Ellis-Van Creveld Syndrome, Encephalitis, Enchondromatosis, Epidermal Necrolysis, Toxic, Facial Hemiatrophy, Factor XII Deficiency, Fanconi Anemia, Felty's Syndrome, Fibrous Dysplasia, Polyostotic, Fox-Fordyce Disease, Friedreich Ataxia, Fusobacterium, Gardner Syndrome, Gaucher Disease, Gerstmann Syndrome, Giant Lymph Node Hyperplasia, Glycogen Storage Disease Type I, Glycogen Storage Disease Type II, Glycogen Storage Disease Type IV, Glycogen Storage Disease Type V, Glycogen Storage Disease Type VII, Goldenhar Syndrome, Guillain-Barre Syndrome, Hallermann's Syndrome, Hamartoma Syndrome, Multiple, Hartnup Disease, Hepatolenticular Degeneration, Hepatolenticular Degeneration, Hereditary Sensory and Motor Neuropathy, Hirschsprung Disease, Histiocytic Necrotizing Lymphadenitis, Histiocytosis, Langerhans-Cell, Hodgkin Disease, Homer Syndrome, Huntington Disease, Hyperaldosteronism, Hyperhidrosis, Hyperostosis, Diffuse Idiopathic Skeletal, Hypopituitarism, Inappropriate ADH Syndrome, Intestinal Polyps, Isaacs Syndrome, Kartagener Syndrome, Kearns-Sayre Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay-Weber Syndrome, Kluver-Bucy Syndrome, Korsakoff Syndrome, Lafora Disease, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Langer-Giedion Syndrome, Leigh Disease, Lesch-Nyhan Syndrome, Leukodystrophy, Globoid Cell, Li-Fraumeni Syndrome, Long QT Syndrome, Machado-Joseph Disease, Mallory-Weiss Syndrome, Marek Disease, Marfan Syndrome, Meckel Diverticulum, Meige Syndrome, Melkersson-Rosenthal Syndrome, Meniere Disease, Mikulicz's Disease, Miller Fisher Syndrome, Mobius Syndrome, Moyamoya Disease, Mucocutaneous Lymph Node Syndrome, Mucopolysaccharidosis I, Mucopolysaccharidosis II, Mucopolysaccharidosis III, Mucopolysaccharidosis IV, Mucopolysaccharidosis VI, Multiple Endocrine Neoplasia Type 1, Munchausen Syndrome by Proxy, Muscular Atrophy, Spinal, Narcolepsy, Neuroaxonal Dystrophies, Neuromyelitis Optica, Neuronal Ceroid-Lipofuscinoses, Niemann-Pick Diseases, Noonan Syndrome, Optic Atrophies, Hereditary, Osteitis Deformans, Osteochondritis, Osteochondrodysplasias, Osteolysis, Essential, Paget Disease Extramammary, Paget's Disease, Mammary, Panniculitis, Nodular Nonsuppurative, Papillon-Lefevre Disease, Paralysis, Pelizaeus-Merzbacher Disease, Pemphigus, Benign Familial, Penile Induration, Pericarditis, Constrictive, Peroxisomal Disorders, Peutz-Jeghers Syndrome, Pick Disease of the Brain, Pierre Robin Syndrome, Pigmentation Disorders, Pityriasis Lichenoides, Polycystic Ovary Syndrome, Polyendocrinopathies, Autoimmune, Prader-Willi Syndrome, Pupil Disorders, Rett Syndrome, Reye Syndrome, Rubinstein-Taybi Syndrome, Sandhoff Disease, Sarcoma, Ewing's, Schnitzler Syndrome, Sjogren's Syndrome, Sjogren-Larsson Syndrome, Smith-Lemli-Opitz Syndrome, Spinal Muscular Atrophies of Childhood, Sturge-Weber Syndrome, Sweating, Gustatory, Takayasu Arteritis, Tangier Disease, Tay-Sachs Disease, Thromboangiitis Obliterans, Thyroiditis, Autoimmune, Tietze's Syndrome, Togaviridae Infections, Tolosa-Hunt Syndrome, Tourette Syndrome, Uveomeningoencephalitic Syndrome, Waardenburg's Syndrome, Wegener Granulomatosis, Weil Disease, Werner Syndrome, Williams Syndrome, Wilms Tumor, Wolff-Parkinson-White Syndrome, Wolfram Syndrome, Wolman Disease, Zellweger Syndrome, Zollinger-Ellison Syndrome, and von Willebrand Diseases.

Various autoimmune diseases and autoimmune-related diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As used herein, the term “autoimmune disease” refers to a disease in which the body produces antibodies that attack its own tissues. As a non-limiting example, the autoimmune disease may be Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticaria, Axonal & neuronal neuropathies, Balo disease, Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia**, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's Granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosis, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo, and Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis (GPA).

Various kidney diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the kidney disease Abderhalden-Kaufmann-Lignac syndrome (Nephropathic Cystinosis), Abdominal Compartment Syndrome, Acute Kidney Failure/Acute Kidney Injury, Acute Lobar Nephronia, Acute Phosphate Nephropathy, Acute Tubular Necrosis, Adenine Phosphoribosyltransferase Deficiency, Adenovirus Nephritis, Alport Syndrome, Amyloidosis, ANCA Vasculitis Related to Endocarditis and Other Infections, Angiomyolipoma, Analgesic Nephropathy, Anorexia Nervosa and Kidney Disease, Angiotensin Antibodies and Focal Segmental Glomerulosclerosis, Antiphospholipid Syndrome, Anti-TNF-α Therapy-related Glomerulonephritis, APOL1 Mutations, Apparent Mineralocorticoid Excess Syndrome, Aristolochic Acid Nephropathy, Chinese Herbal Nephropathy, Balkan Endemic Nephropathy, Bartter Syndrome, Beeturia, β-Thalassemia Renal Disease, Bile Cast Nephropathy, BK Polyoma Virus Nephropathy in the Native Kidney, Bladder Rupture, Bladder Sphincter Dyssynergia, Bladder Tamponade, Border-Crossers' Nephropathy, Bourbon Virus and Acute Kidney Injury, Burnt Sugarcane Harvesting and Acute Renal Dysfunction, Byetta and Renal Failure, C1q Nephropathy, Cannabinoid Hyperemesis Acute Renal Failure, Cardiorenal syndrome, Carfilzomib-Indiced Renal Injury, CFHR5 nephropathy, Charcot-Marie-Tooth Disease with Glomerulopathy, Cherry Concentrate and Acute Kidney Injury, Cholesterol Emboli, Churg-Strauss syndrome, Chyluria, Colistin Nephrotoxicity, Collagenofibrotic Glomerulopathy, Collapsing Glomerulopathy, Collapsing Glomerulopathy Related to CMV, Congenital Nephrotic Syndrome, Conorenal syndrome (Mainzer-Saldino Syndrome or Saldino-Mainzer Disease), Contrast Nephropathy, Copper Sulfate Intoxication, Cortical Necrosis, Crizotinib-related Acute Kidney Injury, Cryoglobuinemia, Crystalglobulin-Induced Nephropathy, Crystal-Induced Acute Kidney injury, Cystic Kidney Disease, Acquired, Cystinuria, Dasatinib-Induced Nephrotic-Range Proteinuria, Dense Deposit Disease (MPGN Type 2), Dent Disease (X-linked Recessive Nephrolithiasis), Dialysis Disequilibrium Syndrome, Diabetes and Diabetic Kidney Disease, Diabetes Insipidus, Dietary Supplements and Renal Failure, Drugs of Abuse and Kidney Disease, Duplicated Ureter, EAST syndrome, Ebola and the Kidney, Ectopic Kidney, Ectopic Ureter, Edema, Swelling, Erdheim-Chester Disease, Fabry's Disease, Familial Hypocalciuric Hypercalcemia, Fanconi Syndrome, Fraser syndrome, Fibronectin Glomerulopathy, Fibrillary Glomerulonephritis and Immunotactoid Glomerulopathy, Fraley syndrome, Focal Segmental Glomerulosclerosis, Focal Sclerosis, Focal Glomerulosclerosis, Galloway Mowat syndrome, Giant Cell (Temporal) Arteritis with Kidney Involvement, Gestational Hypertension, Gitelman Syndrome, Glomerular Diseases, Glomerular Tubular Reflux, Glycosuria, Goodpasture Syndrome, Hair Dye Ingestion and Acute Kidney Injury, Hantavirus Infection Podocytopathy, Hematuria (Blood in Urine), Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Hemophagocytic Syndrome, Hemorrhagic Cystitis, Hemorrhagic Fever with Renal Syndrome (HFRS, Hantavirus Renal Disease, Korean Hemorrhagic Fever, Epidemic Hemorrhagic Fever, Nephropathis Epidemica), Hemosiderosis related to Paroxysmal Nocturnal Hemoglobinuria and Hemolytic Anemia, Hepatic Glomerulopathy, Hepatic Veno-Occlusive Disease, Sinusoidal Obstruction Syndrome, Hepatitis C-Associated Renal Disease, Hepatorenal Syndrome, Herbal Supplements and Kidney Disease, High Blood Pressure and Kidney Disease, HIV-Associated Nephropathy (HIVAN), Horseshoe Kidney (Renal Fusion), Hunner's Ulcer, Hyperaldosteronism, Hypercalcemia, Hyperkalemia, Hypermagnesemia, Hypernatremia, Hyperoxaluria, Hyperphosphatemia, Hypocalcemia, Hypokalemia, Hypokalemia-induced renal dysfunction, Hypokalemic Periodic Paralysis, Hypomagnesemia, Hyponatremia, Hypophosphatemia, IgA Nephropathy, IgG4 Nephropathy, Interstitial Cystitis, Painful Bladder Syndrome (Questionnaire), Interstitial Nephritis, Ivemark's syndrome, Ketamine-Associated Bladder Dysfunction, Kidney Stones, Nephrolithiasis, Kombucha Tea Toxicity, Lead Nephropathy and Lead-Related Nephrotoxicity, Leptospirosis Renal Disease, Light Chain Deposition Disease, Monoclonal Immunoglobulin Deposition Disease, Liddle Syndrome, Lightwood-Albright Syndrome, Lipoprotein Glomerulopathy, Lithium Nephrotoxicity, LMX1B Mutations Cause Hereditary FSGS, Loin Pain Hematuria, Lupus, Systemic Lupus Erythematosis, Lupus Kidney Disease, Lupus Nephritis, Lupus Nephritis with Antineutrophil Cytoplasmic Antibody Seropositivity, Lyme Disease-Associated Glomerulonephritis, Malarial Nephropathy, Malignancy-Associated Renal Disease, Malignant Hypertension, Malakoplakia, Meatal Stenosis, Medullary Cystic Kidney Disease, Medullary Sponge Kidney, Megaureter, Melamine Toxicity and the Kidney, Membranoproliferative Glomerulonephritis, Membranous Nephropathy, MesoAmerican Nephropathy, Metabolic Acidosis, Metabolic Alkalosis, Methotrexate-related Renal Failure, Microscopic Polyangiitis, Milk-alkalai syndrome, Minimal Change Disease, MDMA (Molly; Ecstacy; 3,4-Methylenedioxymethamphetamine) and Kidney Failure, Multicystic dysplastic kidney, Multiple Myeloma, Myeloproliferative Neoplasms and Glomerulopathy, Nail-patella Syndrome, Nephrocalcinosis, Nephrogenic Systemic Fibrosis, Nephroptosis (Floating Kidney, Renal Ptosis), Nephrotic Syndrome, Neurogenic Bladder, Nodular Glomerulosclerosis, Non-Gonococcal Urethritis, Nutcracker syndrome, Orofaciodigital Syndrome, Orotic Aciduria, Orthostatic Hypotension, Orthostatic Proteinuria, Osmotic Diuresis, Ovarian Hyperstimulation Syndrome, Page Kidney, Papillary Necrosis, Papillorenal Syndrome (Renal-Coloboma Syndrome, Isolated Renal Hypoplasia), Parvovirus B19 and the Kidney, The Peritoneal-Renal Syndrome, Posterior Urethral Valve, Post-infectious Glomerulonephritis, Post-streptococcal Glomerulonephritis, Polyarteritis Nodosa, Polycystic Kidney Disease, Posterior Urethral Valves, Preeclampsia, Propofol infusion syndrome, Proliferative Glomerulonephritis with Monoclonal IgG Deposits (Nasr Disease), Propolis (Honeybee Resin) Related Renal Failure, Proteinuria (Protein in Urine), Pseudohyperaldosteronism, Pseudohypobicarbonatemia, Pseudohypoparathyroidism, Pulmonary-Renal Syndrome, Pyelonephritis (Kidney Infection), Pyonephrosis, Radiation Nephropathy, Ranolazine and the Kidney, Refeeding syndrome, Reflux Nephropathy, Rapidly Progressive Glomerulonephritis, Renal Abscess, Perinephric Abscess, Renal Agenesis, Renal Arcuate Vein Microthrombi-Associated Acute Kidney Injury, Renal Artery Aneurysm, Renal Artery Stenosis, Renal Cell Cancer, Renal Cyst, Renal Hypouricemia with Exercise-induced Acute Renal Failure, Renal Infarction, Renal Osteodystrophy, Renal Tubular Acidosis, Renin Secreting Tumors (Juxtaglomerular Cell Tumor), Reset Osmostat, Retrocaval Ureter, Retroperitoneal Fibrosis, Rhabdomyolysis, Rhabdomyolysis related to Bariatric Surgery, Rheumatoid Arthritis-Associated Renal Disease, Sarcoidosis Renal Disease, Salt Wasting, Renal and Cerebral, Schistosomiasis and Glomerular Disease, Schimke immuno-osseous dysplasia, Scleroderma Renal Crisis, Serpentine Fibula-Polycystic Kidney Syndrome, Exner Syndrome, Sickle Cell Nephropathy, Silica Exposure and Chronic Kidney Disease, Sri Lankan Farmers' Kidney Disease, Sjögren's Syndrome and Renal Disease, Synthetic Cannabinoid Use and Acute Kidney Injury, Kidney Disease Following Hematopoietic Cell Transplantation, Kidney Disease Related to Stem Cell Transplantation, Thin Basement Membrane Disease, Benign Familial Hematuria, Trigonitis, Tuberculosis, Genitourinary, Tuberous Sclerosis, Tubular Dysgenesis, Immune Complex Tubulointerstitial Nephritis Due to Autoantibodies to the Proximal Tubule Brush Border, Tumor Lysis Syndrome, Uremia, Uremic Optic Neuropathy, Ureteritis Cystica, Ureterocele, Urethral Caruncle, Urethral Stricture, Urinary Incontinence, Urinary Tract Infection, Urinary Tract Obstruction, Vesicointestinal Fistula, Vesicoureteral Reflux, Volatile Anesthetics and Acute Kidney Injury, Von Hippel-Lindau Disease, Waldenstrom's Macroglobulinemic Glomerulonephritis, Warfarin-Related Nephropathy, Wasp Stings and Acute Kidney Injury, Wegener's Granulomatosis, Granulomatosis with Polyangiitis, West Nile Virus and Chronic Kidney Disease, and Wunderlich syndrome.

Various cardiovascular diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the cardiovascular disease may be Ischemic heart disease also known as coronary artery disease, Cerebrovascular disease (Stroke), Peripheral vascular disease, Heart failure, Rheumatic heart disease, and Congenital heart disease.

Various antibody deficiencies may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the antibody deficiencies may be X-Linked Agammaglobulinemia (XLA), Autosomal Recessive Agammaglobulinemia (ARA), Common Variable Immune Deficiency (CVID), IgG (IgG1, IgG2, IgG3 and IgG4) Subclass Deficiency, Selective IgA Deficiency, Specific Antibody Deficiency (SAD), Transient Hypogammaglobulinemia of Infancy, Antibody Deficiency with Normal or Elevated Immunoglobulins, Selective IgM Deficiency, Immunodeficiency with Thymoma (Good's Syndrome), Transcobalamin II Deficiency, Warts, Hypogammaglobulinemia, Infection, Myelokathexis (WHIM) Syndrome, Drug-Induced Antibody Deficiency, Kappa Chain Deficiency, Heavy Chain Deficiencies, Post-Meiotic Segregation (PMS2) Disorder, and Unspecified Hypogammaglobulinemia.

Various ocular diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the ocular disease may be thyroid eye disease (TED), Graves' disease (GD) and orbitopathy, Retina Degeneration, Cataract, optic atrophy, macular degeneration, Leber congenital amaurosis, retinal degeneration, cone-rod dystrophy, Usher syndrome, leopard syndrome, photophobia, and photoaversion.

Various neurological diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the neurological disease may be Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS—Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain and Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavernous Malformation, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Chiari Malformation, Cholesterol Ester Storage Disease, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia-Multi-Infarct, Dementia-Semantic, Dementia-Subcortical, Dementia With Lewy Bodies, Dentate Cerebellar Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia Cerebellaris Myoclonica, Dyssynergia Cerebellaris Progressiva, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles, Encephalopathy, Encephalopathy (familial infantile), Encephalotrigeminal Angiomatosis, Epilepsy, Epileptic Hemiplegia, Erb's Palsy, Erb-Duchenne and Dej erine-Klumpke Palsies, Essential Tremor, Extrapontine Myelinolysis, Fabry Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralyses, Familial Spastic Paralysis, Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, Fisher Syndrome, Floppy Infant Syndrome, Foot Drop, Friedreich's Ataxia, Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage Disease, Guillain-Barré Syndrome, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster, Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome, Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome, Huntington's Disease, Hydranencephaly, Hydrocephalus, Hydrocephalus-Normal Pressure, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaacs' Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Kliiver-Bucy Syndrome, Korsakoffs Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Diseases, Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus-Neurological Sequelae, Lyme Disease—Neurological Complications, Machado-Joseph Disease, Macrocephaly, Megalocephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke, Mitochondrial Myopathy, Moebius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidosis, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy, Myopathy-Congenital, Myopathy-Thyrotoxic, Myotonia, Myotonia Congenita, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Complications of Lyme Disease, Neurological Consequences of Cytomegalovirus Infection, Neurological Manifestations of Pompe Disease, Neurological Sequelae Of Lupus, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy-Hereditary, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain—Chronic, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick's Disease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Post infectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Dentatum Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen's Encephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease, Refsum Disease—Infantile, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seitelberger Disease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjögren's Syndrome, Sleep Apnea, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Spinocerebellar Degeneration, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Short-lasting, Unilateral, Neuralgiform (SUNCT) Headache, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen's Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of the Central and Peripheral Nervous Systems, Von Economo's Disease, Von Hippel-Lindau Disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple's Disease, Williams Syndrome, Wilson Disease, Wolman's Disease, X-Linked Spinal and Bulbar Muscular Atrophy.

Various psychological disorders may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the psychological disorders may be Aboulia, Absence epilepsy, Acute stress Disorder, Adjustment Disorders, Adverse effects of medication NOS, Age related cognitive decline, Agoraphobia, Alcohol Addiction, Alzheimer's Disease, Amnesia (also known as Amnestic Disorder), Amphetamine Addiction, Anorexia Nervosa, Anterograde amnesia, Antisocial personality disorder (also known as Sociopathy), Anxiety Disorder (Also known as Generalized Anxiety Disorder), Anxiolytic related disorders, Asperger's Syndrome (now part of Autism Spectrum Disorder), Attention Deficit Disorder (Also known as ADD), Attention Deficit Hyperactivity Disorder (Also known as ADHD), Autism Spectrum Disorder (also known as Autism), Autophagia, Avoidant Personality Disorder, Barbiturate related disorders, Benzodiazepine related disorders, Bereavement, Bibliomania, Binge Eating Disorder, Bipolar disorder (also known as Manic Depression, includes Bipolar I and Bipolar II), Body Dysmorphic Disorder, Borderline intellectual functioning, Borderline Personality Disorder, Breathing-Related Sleep Disorder, Brief Psychotic Disorder, Bruxism, Bulimia Nervosa, Caffeine Addiction, Cannabis Addiction, Catatonic disorder, Catatonic schizophrenia, Childhood amnesia, Childhood Disintegrative Disorder (now part of Autism Spectrum Disorder), Childhood Onset Fluency Disorder (formerly known as Stuttering), Circadian Rhythm Disorders, Claustrophobia, Cocaine related disorders, Communication disorder, Conduct Disorder, Conversion Disorder, Cotard delusion, Cyclothymia (also known as Cyclothymic Disorder), Delerium, Delusional Disorder, dementia, Dependent Personality Disorder (also known as Asthenic Personality Disorder), Depersonalization disorder (now known as Depersonalization/Derealization Disorder), Depression (also known as Major Depressive Disorder), Depressive personality disorder, Derealization disorder (now known as Depersonalization/Derealization Disorder), Dermotillomania, Desynchronosis, Developmental coordination disorder, Diogenes Syndrome, Disorder of written expression, Dispareunia, Dissocial Personality Disorder, Dissociative Amnesia, Dissociative Fugue, Dissociative Identity Disorder (formerly known as Multiple Personality Disorder), Down syndrome, Dyslexia, Dyspareunia, Dysthymia (now known as Persistent Depressive Disorder), Eating disorder NOS, Ekbom's Syndrome (Delusional Parasitosis), Emotionally unstable personality disorder, Encopresis, Enuresis (bedwetting), Erotomania, Exhibitionistic Disorder, Expressive language disorder, Factitious Disorder, Female Sexual Disorders, Fetishistic Disorder, Folie à deux, Fregoli delusion, Frotteuristic Disorder, Fugue State, Ganser syndrome, Gambling Addiction, Gender Dysphoria (formerly known as Gender Identity Disorder), Generalized Anxiety Disorder, General adaptation syndrome, Grandiose delusions, Hallucinogen Addiction, Haltlose personality disorder, Histrionic Personality Disorder, Primary hypersomnia, Huntington's Disease, Hypoactive sexual desire disorder, Hypochondriasis, Hypomania, Hyperkinetic syndrome, Hypersomnia, Hysteria, Impulse control disorder, Impulse control disorder NOS, Inhalant Addiction, Insomnia, Intellectual Development Disorder, Intermittent Explosive Disorder, Joubert syndrome, Kleptomania, Korsakoff's syndrome, Lacunar amnesia, Language Disorder, Learning Disorders, Major Depression (also known as Major Depressive Disorder), major depressive disorder, Male Sexual Disorders, Malingering, Mathematics disorder, Medication-related disorder, Melancholia, Mental Retardation (now known as Intellectual Development Disorder), Misophonia, Morbid jealousy, Multiple Personality Disorder (now known as Dissociative Identity Disorder), Munchausen Syndrome, Munchausen by Proxy, Narcissistic Personality Disorder, Narcolepsy, Neglect of child, Neurocognitive Disorder (formerly known as Dementia), Neuroleptic-related disorder, Nightmare Disorder, Non Rapid Eye Movement, Obsessive-Compulsive Disorder, Obsessive-Compulsive Personality Disorder (also known as Anankastic Personality Disorder), Oneirophrenia, Onychophagia, Opioid Addiction, Oppositional Defiant Disorder, Orthorexia (ON), Pain disorder, Panic attacks, Panic Disorder, Paranoid Personality Disorder, Parkinson's Disease, Partner relational problem, Passive-aggressive personality disorder, Pathological gambling, Pedophilic Disorder, Perfectionism, Persecutory delusion, Persistent Depressive Disorder (also known as Dysthymia), Personality change due to a general medical condition, Personality disorder, Pervasive developmental disorder (PDD), Phencyclidine related disorder, Phobic disorder, Phonological disorder, Physical abuse, Pica, Polysubstance related disorder, Postpartum Depression, Post-traumatic embitterment disorder (PTED), Post-Traumatic Stress Disorder, Premature ejaculation, Premenstrual Dysphoric Disorder, Psychogenic amnesia, Psychological factor affecting medical condition, Psychoneurotic personality disorder, Psychotic disorder, not otherwise specified, Pyromania, Reactive Attachment Disorder, Reading disorder, Recurrent brief depression, Relational disorder, REM Sleep Behavior Disorder, Restless Leg Syndrome, Retrograde amnesia, Retts Disorder (now part of Autism Spectrum Disorder), Rumination syndrome, Sadistic personality disorder, Schizoaffective Disorder, Schizoid Personality Disorder, Schizophrenia, Schizophreniform disorder, Schizotypal Personality Disorder, Seasonal Affective Disorder, Sedative, Hypnotic, or Anxiolytic Addiction, Selective Mutism, Self-defeating personality disorder, Separation Anxiety Disorder, Sexual Disorders Female, Sexual Disorders Male, Sexual Addiction, Sexual Masochism Disorder, Sexual Sadism Disorder, Shared Psychotic Disorder, Sleep Arousal Disorders, Sleep Paralysis, Sleep Terror Disorder (now part of Nightmare Disorder, Social Anxiety Disorder, Somatization Disorder, Specific Phobias, Stendhal syndrome, Stereotypic movement disorder, Stimulant Addiction, Stuttering (now known as Childhood Onset Fluency Disorder), Substance related disorder, Tardive dyskinesia, Tobacco Addiction, Tourettes Syndrome, Transient tic disorder, Transient global amnesia, Transvestic Disorder, Trichotillomania, Undifferentiated Somatoform Disorder, Vaginismus, and Voyeuristic Disorder.

Various lung diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the lung diseases may be Asbestosis, Asthma, Bronchiectasis, Bronchitis, Chronic Cough, Chronic Obstructive Pulmonary Disease (COPD), Croup, Cystic Fibrosis, Hantavirus, Idiopathic Pulmonary Fibrosis, Pertussis, Pleurisy, Pneumonia, Pulmonary Embolism, Pulmonary Hypertension, Sarcoidosis, Sleep Apnea, Spirometry, Sudden Infant Death Syndrome (SIDS), Tuberculosis, Alagille Syndrome, Autoimmune Hepatitis, Biliary Atresia, Cirrhosis, ERCP (Endoscopic Retrograde Cholangiopancreatography), and Hemochromatosis. Nonalcoholic Steatohepatitis, Porphyria, Primary Biliary Cirrhosis, Primary Sclerosing Cholangitis.

Various bone diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the bone diseases may be osteoporosis, neurofibromatosis, osteogenesis imperfecta (OI), rickets, osteosarcoma, achondroplasia, fracture, osteomyelitis, Ewing tumor of bone, osteomalacia, hip dysplasia, Paget disease of bone, marble bone disease, osteochondroma, bone cancer, bone disease, osteochondrosis, osteoma, fibrous dysplasia, cleidocranial dysostosis, osteoclastoma, bone cyst, metabolic bone disease, melorheostosis, callus, Caffey syndrome, and mandibulofacial dysostosis.

Various blood diseases may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As a non-limiting example, the blood diseases may be Anemia and CKD (for health care professionals), Aplastic Anemia and Myelodysplastic Syndromes, Deep Vein Thrombosis, Hemochromatosis, Hemophilia, Henoch-Schönlein Purpura, Idiopathic Thrombocytopenic Purpura, Iron-Deficiency Anemia, Pernicious Anemia, Pulmonary Embolism, Sickle Cell Anemia, Sickle Cell Trait and Other Hemoglobinopathies, Thalassemia, Thrombotic Thrombocytopenic Purpura, and Von Willebrand Disease.

In some embodiment, biocircuits of the invention may be used for the treatment of infectious diseases. Biocircuits of the invention may be introduced in cells suitable for adoptive cell transfer such as macrophages, dendritic cells, natural killer cells, and or T cells. Infectious diseases treated by the biocircuits of the invention may be diseases caused by viruses, bacteria, fungi, and/or parasites. IL15-IL15Ra payloads of the invention may be used to increase immune cell proliferation and/or persistence of the immune cells useful in treating infectious diseases.

6. Microbiome

Alterations in the composition of the microbiome may impact the action of anti-cancer therapies. A diverse community of symbiotic, commensal and pathogenic microorganisms exist in all environmentally exposed sites in the body and is herein referred to as the “Microbiome.” Environmentally exposed sites of the body that may be inhabited by a microbiome include the skin, nasopharynx, the oral cavity, respiratory tract, gastrointestinal tract, and the reproductive tract.

In some embodiments, microbiome engineered with the biocircuits of the present invention may be used to improve the efficacy of the anti-cancer immunotherapies. Sivan et al., found that mice with Bifidobacterium in their gut microbiome were more responsive to immune check point blockage e.g. anti PD-L1 immunotherapy in subcutaneous melanoma tumor model (Sivan A., et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science 2015; 350:1084-9; the contents of which are incorporated herein by reference in their entirety). In one embodiment, protein, RNA and/or other biomolecules derived from the microbiome may be used as a payload to influence the efficacy of the anti-cancer immunotherapies. In other embodiments, the microorganisms may be delivered along with immunotherapeutic compositions of the present invention to improve the efficacy of immunotherapy.

7. Tools and Agents for Making Therapeutics

Provided in the present invention are tools and agents that may be used in generating immunotherapeutics for reducing a tumor volume or burden in a subject in need. A considerable number of variables are involved in producing a therapeutic agent, such as structure of the payload, type of cells, method of gene transfers, method and time of ex vivo expansion, pre-conditioning and the amount and type of tumor burden in the subject. Such parameters may be optimized using tools and agents described herein.

Cell Lines

The present disclosure provides a mammalian cell that has been genetically modified with the compositions of the invention. Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include, but are not limited to Human embryonic kidney cell line 293, fibroblast cell line NIH 3T3, human colorectal carcinoma cell line HCT116, ovarian carcinoma cell line SKOV-3, immortalized T cell lines Jurkat cells, lymphoma cell line Raji cells, NALM-6 cells, K562 cells, HeLa cells, PC12 cells, HL-60 cells, NK cell lines (e.g. NKL. NK962, and YTS), and the like. In some instances, the cell is not an immortalized cell line, but instead a cell obtained from an individual and is herein referred to as a primary cell. For example, the cell is a T lymphocyte obtained from an individual. Other examples include, but are not limited to cytotoxic cells, stem cells, peripheral blood mononuclear cells or progenitor cells obtained from an individual.

Tracking SREs, Biocircuits and Cell Lines

In some embodiments, it may be desirable to track the compositions of the invention or the cells modified by the compositions of the invention. Tracking may be achieved by using payloads such as reporter moieties, which, as used herein, refers to any protein capable of creating a detectable signal, in response to an input. Examples include alkaline phosphatase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, peroxidase, β-lactamase, catalytic antibodies, bioluminescent proteins e.g. luciferase, and fluorescent proteins such as Green fluorescent protein (GFP).

Reporter moieties may be used to monitor the response of the DD upon addition of the ligand corresponding to the DD. In other instances, reporter moieties may be used to track cell survival, persistence, cell growth, and/or localization in vitro, in vivo, or ex vivo.

In some embodiments, the preferred reporter moiety may be luciferase proteins. In one embodiment, the reporter moiety is the Renilla luciferase, or a firefly luciferase.

Animal Models

The utility and efficacy of the compositions of the present invention may be tested in vivo animal models, preferably mouse models. Mouse models used to may be syngeneic mouse models wherein mouse cells are modified with compositions of the invention and tested in mice of the same genetic background. Examples include pMEL-1 and 4T1 mouse models. Alternatively, xenograft models where human cells such as tumor cells and immune cells are introduced into immunodeficient mice may also be utilized in such studies. Immunodeficient mice used may be CByJ.Cg-Foxn1^(nu)/J, B6; 129S7-Rag1^(tm1Mom)/J, B6.129S7-Rag1^(tm1mom)/J, B6. CB17-Prkdc^(scid)/SzJ, NOD.129S7 (B6)-Rag1^(tm1Mom)/J, NOD.Cg-Rag1^(th1Mom)Prf1^(tm1Sd)z/Sz, NOD.CB17-Prkdc^(scid)/SzJ, NOD.Cg-Prkdc^(scid)B2m^(tm1Unc)/J NOD-scid IL2Rg^(null), Nude (nu) mice, SCID mice, NOD mice, RAG1/RAG2 mice, NOD-Scid mice, IL2rgnull mice, b2mnull mice, NOD-scid IL2rγnull mice, NOD-scid-B2mnull mice, and HLA transgenic mice.

Cellular Assays

In some embodiments, the effectiveness of the compositions of the inventions as immunotherapeutic agents may be evaluated using cellular assays. Levels of expression and/or identity of the compositions of the invention may be determined according to any methods known in the art for identifying proteins and/or quantitating proteins levels. In some embodiments, such methods may include Western Blotting, flow cytometry, and immunoprecipitation.

Provided herein are methods for functionally characterizing cells expressing SRE, biocircuits and compositions of the invention. In some embodiments, functional characterization is carried out in primary immune cells or immortalized immune cell lines and may be determined by expression of cell surface markers. Examples of cell surface markers for T cells include, but are not limited to, CD3, CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 and HLA-DR, CD 69, CD28, CD44, IFNgamma. Examples of cell surface markers for antigen presenting cells include, but are not limited to, MHC class I, MEW Class II, CD40, CD45, B7-1, B7-2, IFN γ receptor and IL2 receptor, ICAM-1 and/or Fcγ receptor. Examples of cell surface markers for dendritic cells include, but are not limited to, MEW class I, MHC Class II, B7-2, CD18, CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR and/or Dectin-1 and the like; while in some cases also having the absence of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD 19, CD20, CD56, and/or CD57. Examples of cell surface markers for NK cells include, but are not limited to, CCL3, CCL4, CCL5, Granulysin, Granzyme B, Granzyme K, IL10, IL22, IFNg, LAP, Perforin, and TNFa.

8. Gene Editing

The CRISPR-Cas9 system is a novel genome editing system which has been rapidly developed and implemented in a multitude of model organisms and cell types, and supplants other genome editing technologies, such as TALENs and ZFNs. CRISPRs are sequence motifs are present in bacterial and archaeal genomes, and are composed of short (about 24-48 nucleotide) direct repeats separated by similarly sized, unique spacers (Grissa et al. BMC Bioinformatics 8, 172 (2007). They are generally flanked by a set of CRISPR-associated (Cas) protein-coding genes that are required for CRISPR maintenance and function (Barrangou et al., Science 315, 1709 (2007), Brouns et al., Science 321, 960 (2008), Haft et al. PLoS Comput Biol 1, e60 (2005)). CRISPR-Cas systems provide adaptive immunity against invasive genetic elements (e.g., viruses, phages and plasmids) (Horvath and Barrangou, Science, 2010, 327: 167-170; Bhaya et al., Annu. Rev. Genet., 2011, 45: 273-297; and Brrangou R, RNA, 2013, 4: 267-278). Three different types of CRISPR-Cas systems have been classified in bacteria and the type II CRISPR-Cas system is most studied. In the bacterial Type II CRISPR-Cas system, small CRISPR RNAs (crRNAs) processed from the pre-repeat-spacer transcript (pre-crRNA) in the presence of a trans-activating RNA (tracrRNA)/Cas9 can form a duplex with the tracrRNA/Cas9 complex. The mature complex is recruited to a target double strand DNA sequence that is complementary to the spacer sequence in the tracrRNA: crRNA duplex to cleave the target DNA by Cas9 endonuclease (Garneau et al., Nature, 2010, 468: 67-71; Jinek et al., Science, 2012, 337: 816-821; Gasiunas et al., Proc. Natl Acad. Sci. USA., 109: E2579-2586; and Haurwitz et al., Science, 2010, 329: 1355-1358). Target recognition and cleavage by the crRNA: tracrRNA/Cas9 complex in the type II CRISPR-CAS system not only requires a sequence in the tracrRNA: crRNA duplex that is a complementary to the target sequence (also called “protospacer” sequence) but also requires a protospacer adjacent motif (PAM) sequence located 3′end of the protospacer sequence of a target polynucleotide. The PAM motif can vary between different CRISPR-Cas systems.

CRISPR-Cas9 systems have been developed and modified for use in genetic editing and prove to be a high effective and specific technology for editing a nucleic acid sequence even in eukaryotic cells. Many researchers disclosed various modifications to the bacterial CRISPR-Cas systems and demonstrated that CRISPR-Cas systems can be used to manipulate a nucleic acid in a cell, such as in a mammalian cell and in a plant cell. Representative references include U.S. Pat. Nos. 8,993,233; 8,999,641; 8,945,839; 8,932,814; 8,906, 616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,771,945; and 8,697,359; US patent publication NOs.: 20150031134; 20150203872; 20150218253; 20150176013; 20150191744; 20150071889; 20150067922; and 20150167000; each of which is incorporated herein by reference in their entirety.

However, controlling the effects and activity of the CRISPR-Cas system (e.g., guide RNA and nuclease) has been challenging and often can be problematic.

The biocircuits of the present invention and/or any of their components may be utilized in regulating or tuning the CRISPR/Cas9 system to optimize its utility.

In some embodiments, the payloads of the effector modules of the invention may include alternative isoforms or orthologs of the Cas9 enzyme.

The most commonly used Cas9 is derived from Streptococcus pyogenes and the RuvC domain can be inactivated by a D10A mutation and the HNH domain can be inactivated by an H840A mutation.

In addition to Cas9 derived from S. pyogenes, other RNA guided endonucleases (RGEN) may also be used for programmable genome editing. Cas9 sequences have been identified in more than 600 bacterial strains. Though Cas9 family shows high diversity of amino acid sequences and protein sizes, All Cas9 proteins share a common architecture with a central HNH nuclease domain and a split RuvC/RHase H domain. Examples of Cas9 orthologs from other bacterial strains including but not limited to, Cas proteins identified in Acaryochloris marina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifex degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp. bulgaricus PB2003/044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas sp. J5666; Pseudoalteromonas haloplanktis TAC125; Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737).

In addition to Cas9 orthologs, other Cas9 variants such as fusion proteins of inactive dCas9 and effector domains with different functions may be served as a platform for genetic modulation. Any of the foregoing enzymes may be useful in the present invention.

9. Stem Cell Applications

The biocircuits of the present invention and/or any of their components may be utilized in the regulated reprogramming of cells, stem cell engraftment or other application where controlled or tunable expression of such reprogramming factors are useful.

The biocircuits of the present invention may be used in reprogramming cells including stem cells or induced stem cells. Induction of induced pluripotent stem cells (iPSC) was first achieved by Takahashi and Yamanaka (Cell, 2006. 126(4):663-76; herein incorporated by reference in its entirety) using viral vectors to express KLF4, c-MYC, OCT4 and SOX2 otherwise collectively known as KMOS.

Excisable lentiviral and transposon vectors, repeated application of transient plasmid, episomal and adenovirus vectors have also been used to try to derive iPSC (Chang, C.-W., et al., Stem Cells, 2009. 27(5):1042-1049; Kaji, K., et al., Nature, 2009. 458(7239):771-5; Okita, K., et al., Science, 2008. 322(5903):949-53; Stadtfeld, M., et al., Science, 2008. 322(5903):945-9; Woltjen, K., et al., Nature, 2009; Yu, J., et al., Science, 2009:1172482; Fusaki, N., et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009. 85(8):348-62; each of which is herein incorporated by reference in its entirety).

DNA-free methods to generate human iPSC has also been derived using serial protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties (Kim, D., et al., Cell Stem Cell, 2009. 4(6): 472-476; Zhou, H., et al., Cell Stem Cell, 2009. 4(5):381-4; each of which is herein incorporated by reference in its entirety), and infectious transgene delivery using the Sendai virus (Fusaki, N., et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009. 85 (8): p. 348-62; herein incorporated by reference in its entirety).

The effector modules of the present invention may include a payload comprising any of the genes including, but not limited to, OCT such as OCT4, SOX such as SOX1, SOX2, SOX3, SOX15 and SOX18, NANOG, KLF such as KLF1, KLF2, KLF4 and KLF5, MYC such as c-MYC and n-MYC, REM2, TERT and LIN28 and variants thereof in support of reprogramming cells. Sequences of such reprogramming factors are taught in for example International Application PCT/US2013/074560, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the payload of the present invention may be cardiac lineage specification factors such as eomesodermin (EOMES), a T-box transcription factor; WNT signaling pathway components such as WNT3 and WNT 3A. EOMES is crucially required for the development of the heart. Cardiomyocyte programming by EOMES involves autocrine activation of the canonical WNT signaling pathway and vice versa. Under conditions that are conducive to promoting cardiac lineage, WNT signaling activates EOMES and EOMES in turn promotes WNT signaling creating a self-sustaining loop that promotes the cardiac lineage. An activation loop that is too weak or too strong promotes non-cardiac fates such as endodermal and other mesodermal fates respectively. The DDs of the present invention may be used to tune EOMES and WNT payload levels to generate an activation loop that initiate and/or sustain cardiac specification during gastrulation.

VII. Definitions

At various places in the present specification, features or functions of the compositions of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual sub-combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.

Activity: As used herein, the term “activity” refers to the condition in which things are happening or being done. Compositions of the invention may have activity and this activity may involve one or more biological events. In some embodiments, biological events may include cell signaling events. In some embodiments, biological events may include cell signaling events associated protein interactions with one or more corresponding proteins, receptors, small molecules or any of the biocircuit components described herein.

Adoptive cell therapy (ACT): The terms “Adoptive cell therapy” or “Adoptive cell transfer”, as used herein, refer to a cell therapy involving in the transfer of cells into a patient, wherein cells may have originated from the patient, or from another individual, and are engineered (altered) before being transferred back into the patient. The therapeutic cells may be derived from the immune system, such as Immune effector cells: CD4+ T cell; CD8+ T cell, Natural Killer cell (NK cell); and B cells and tumor infiltrating lymphocytes (TILs) derived from the resected tumors. Most commonly transferred cells are autologous anti-tumor T cells after ex vivo expansion or manipulation. For example, autologous peripheral blood lymphocytes can be genetically engineered to recognize specific tumor antigens by expressing T-cell receptors (TCR) or chimeric antigen receptor (CAR).

Agent: As used herein, the term “agent” refers to a biological, pharmaceutical, or chemical compound. Non-limiting examples include simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a receptor, and soluble factor.

Agonist: the term “agonist” as used herein, refers to a compound that, in combination with a receptor, can produce a cellular response. An agonist may be a ligand that directly binds to the receptor. Alternatively, an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise resulting in the modification of another compound so that the other compound directly binds to the receptor. An agonist may be referred to as an agonist of a particular receptor or family of receptors, e.g., agonist of a co-stimulatory receptor.

Antagonist: the term “antagonist” as used herein refers to any agent that inhibits or reduces the biological activity of the target(s) it binds.

Antigen: the term “antigen” as used herein is defined as a molecule that provokes an immune response when it is introduced into a subject or produced by a subject such as tumor antigens which arise by the cancer development itself. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells such as cytotoxic T lymphocytes and T helper cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates. In the context of the invention, the terms “antigens of interest” or “desired antigens” refers to those proteins and/or other biomolecules provided herein that are immunospecifically bound or interact with antibodies of the present invention and/or fragments, mutants, variants, and/or alterations thereof described herein. In some embodiments, antigens of interest may comprise any of the polypeptides or payloads or proteins described herein, or fragments or portions thereof.

Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Alkyl: The terms “alkyl”, “alkoxy”, “hydroxyalkyl”, “alkoxy alkyl”, and “alkoxy carbonyl”, as used herein, include both straight and branched chains containing one to twelve carbon atoms, and/or which may or may not be substituted.

Alkenyl: The terms “alkenyl” and “alkynyl” as used herein alone or as part of a larger moiety shall include both straight and branched chains containing two to twelve carbon atoms.

Aryl: The term “aryl” as used herein alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic, bicyclic and tricyclic carbocyclic ring systems having a total of five to fourteen ring members, wherein at least one ring is aromatic and wherein each ring in the system contains 3 to 8 ring members. The term “aryl” may be used interchangeably with the term “aryl ring.”

Aromatic: The term “aromatic” as used herein, refers to an unsaturated hydrocarbon ring structure with delocalized pi electrons. As used herein “aromatic” may refer to monocyclic, bicyclic or polycyclic aromatic compounds.

Aliphatic: The term “aliphatic” or “aliphatic group” as used herein, refers to a straight or branched C1-C8 hydrocarbon chain or a monocyclic C3-C8hydrocarbon or bicyclic C8-C12 hydrocarbon which are fully saturated or that contains one or more units of unsaturation, that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle” or “cycloalkyl”), and that has a single point of attachment to the rest of the molecule wherein any individual ring in said bicyclic ring system has 3-7 members.

Associated with: As used herein, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, mean that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serve as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the “associated” entities remain physically associated.

Autologous: the term “autologous” as used herein is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

Barcode: the term “barcode” as used herein refers to polynucleotide or amino acid sequence that distinguishes one polynucleotide or amino acid from another.

Biocircuit system: As used herein, a “biocircuit” or “biocircuit system” is defined as a circuit within or useful in biologic systems comprising a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces at least one signal or outcome within, between, as an indicator of, or on a biologic system. Biologic systems are generally understood to be any cell, tissue, organ, organ system or organism, whether animal, plant, fungi, bacterial, or viral. It is also understood that biocircuits may be artificial circuits which employ the stimuli or effector modules taught by the present invention and effect signals or outcomes in acellular environments such as with diagnostic, reporter systems, devices, assays or kits. The artificial circuits may be associated with one or more electronic, magnetic, or radioactive components or parts. In the context of the present invention, a biocircuit includes a destabilizing domain (DD) biocircuit system.

Checkpoint factor: As used herein, a checkpoint factor is any moiety or molecule whose function acts at the junction of a process. For example, a checkpoint protein, ligand or receptor may function to stall or accelerate the cell cycle.

Co-stimulatory molecule: As used herein, in accordance with its meaning in immune T cell activation, refers to a group of immune cell surface receptor/ligands which engage between T cells and APCs and generate a stimulatory signal in T cells which combines with the stimulatory signal in T cells that results from T cell receptor (TCR) recognition of antigen/MHC complex (pMHC) on APCs

Cytokines: the term “cytokines”, as used herein, refers to a family of small soluble factors with pleiotropic functions that are produced by many cell types that can influence and regulate the function of the immune system.

Delivery: the term “delivery” as used herein refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload. A “delivery agent” refers to any agent which facilitates, at least in part, the in vivo delivery of one or more substances (including, but not limited to compounds and/or compositions of the present invention) to a cell, subject or other biological system cells.

Destabilized: As used herein, the term “destable,” “destabilize,” “destabilizing region” or “destabilizing domain” means a region or molecule that is less stable than a starting, reference, wild-type or native form of the same region or molecule.

Engineered: As used herein, embodiments of the invention are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.

Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or 3′ end processing); (3) translation of an RNA into a polypeptide or protein; (4) folding of a polypeptide or protein; and (5) post-translational modification of a polypeptide or protein.

Feature: As used herein, a “feature” refers to a characteristic, a property, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least a compound and/or composition of the present invention and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein. In some embodiments, a fragment of a protein includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more amino acids. In some embodiments, fragments of an antibody include portions of an antibody.

Functional: As used herein, a “functional” biological molecule is a biological entity with a structure and in a form in which it exhibits a property and/or activity by which it is characterized.

Heterocycle: The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used herein refers to monocyclic, bicyclic or tricyclic ring systems having three to fourteen ring members in which one or more ring members is a heteroatom, wherein each ring in the system contains 3 to 7 ring members and is non-aromatic.

Hotspot: As used herein, a “hotspot” or a “mutational hotspot” refers to an amino acid position in a protein coding gene that is mutated (by substitutions) more frequently relative to elsewhere within the same gene.

Hydrophilic: As used herein, “hydrophilic” refers to a molecule that interacts with or has affinity for water.

Hydrophobic: As used herein, “hydrophobic” refers to a molecule that does not interact or have affinity for water.

Immune cells: the term “an immune cell”, as used herein, refers to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4− CD8− double negative T cell, a T γδ cell, a Tαβ cell, a regulatory T cell, a natural killer cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

Immunotherapy: the term “immunotherapy” as used herein, refers to a type of treatment of a disease that uses immunological tools, such as monoclonal antibodies, receptor-immunoglobulin fusion proteins, vaccines and/or immune cells.

In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

Linker: As used herein, a linker refers to a moiety that connects two or more domains, moieties or entities. In one embodiment, a linker may comprise 10 or more atoms. In a further embodiment, a linker may comprise a group of atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. In some embodiments, a linker may comprise one or more nucleic acids comprising one or more nucleotides. In some embodiments, the linker may comprise an amino acid, peptide, polypeptide or protein. In some embodiments, a moiety bound by a linker may include, but is not limited to an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, a protein complex, a payload (e.g., a therapeutic agent), or a marker (including, but not limited to a chemical, fluorescent, radioactive or bioluminescent marker). The linker can be used for any useful purpose, such as to form multimers or conjugates, as well as to administer a payload, as described herein. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomeric units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers, Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (—S—S—) or an azo bond (—N═N—), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bonds include an amido bond which may be cleaved for example by the use of tris(2-carboxyethyl) phosphine (TCEP), or other reducing agents, and/or photolysis, as well as an ester bond which may be cleaved for example by acidic or basic hydrolysis.

Lipophilic: As used herein, the term “lipophilic” refers to an affinity for lipids or fats.

Metabolite: Metabolites are the intermediate products of metabolic reactions catalyzed by enzymes that naturally occur within cells. This term is usually used to describe small molecules, fragments of larger biomolecules or processed products.

Modified: As used herein, the term “modified” refers to a changed state or structure of a molecule or entity as compared with a parent or reference molecule or entity. Molecules may be modified in many ways including chemically, structurally, and functionally. In some embodiments, compounds and/or compositions of the present invention are modified by the introduction of non-natural amino acids.

Mutation: As used herein, the term “mutation” refers to a change and/or alteration. In some embodiments, mutations may be changes and/or alterations to proteins (including peptides and polypeptides) and/or nucleic acids (including polynucleic acids). In some embodiments, mutations comprise changes and/or alterations to a protein and/or nucleic acid sequence. Such changes and/or alterations may comprise the addition, substitution and or deletion of one or more amino acids (in the case of proteins and/or peptides) and/or nucleotides (in the case of nucleic acids and or polynucleic acids). In some embodiments, wherein mutations comprise the addition and/or substitution of amino acids and/or nucleotides, such additions and/or substitutions may comprise 1 or more amino acid and/or nucleotide residues and may include modified amino acids and/or nucleotides. The resulting construct, molecule or sequence of a mutation, change or alteration may be referred to herein as a mutant.

Neoantigen: the term “neoantigen”, as used herein, refers to a tumor antigen that is present in tumor cells but not normal cells and do not induce deletion of their cognate antigen specific T cells in thymus (i.e., central tolerance). These tumor neoantigens may provide a “foreign” signal, similar to pathogens, to induce an effective immune response needed for cancer immunotherapy. A neoantigen may be restricted to a specific tumor. A neoantigen be a peptide/protein with a missense mutation (missense neoantigen), or a new peptide with long, completely novel stretches of amino acids from novel open reading frames (neoORFs). The neoORFs can be generated in some tumors by out-of-frame insertions or deletions (due to defects in DNA mismatch repair causing microsatellite instability), gene-fusion, read-through mutations in stop codons, or translation of improperly spliced RNA (e.g., Saeterdal et al., Proc Natl Acad Sci USA, 2001, 98: 13255-13260).

Off-target: As used herein, “off target” refers to any unintended effect on any one or more target, gene, cellular transcript, cell, and/or tissue.

Operably linked: As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.

Phenyl: As used herein, “phenyl” refers to a cyclic group of atoms with a formula C6H5.

Payload or payload of interest (POI): the terms “payload” and “payload of interest (POI)”, as used herein, are used interchangeable. A payload of interest (POI) refers to any protein or compound whose function is to be altered. In the context of the present invention, the POI is a component in the immune system, including both innate and adaptive immune systems. Payloads of interest may be a protein, a fusion construct encoding a fusion protein, or non-coding gene, or variant and fragment thereof. Payload of interest may, when amino acid based, may be referred to as a protein of interest.

Pharmaceutically acceptable excipients: the term “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions and having the properties of being substantially nontoxic and non-inflammatory in subjects. In some embodiments, pharmaceutically acceptable excipients are vehicles capable of suspending and/or dissolving active agents. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: Pharmaceutically acceptable salts of the compounds described herein are forms of the disclosed compounds wherein the acid or base moiety is in its salt form (e.g., as generated by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts, for example, from non-toxic inorganic or organic acids. In some embodiments, a pharmaceutically acceptable salt is prepared from a parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety. Pharmaceutically acceptable solvate: The term “pharmaceutically acceptable solvate,” as used herein, refers to a crystalline form of a compound wherein molecules of a suitable solvent are incorporated in the crystal lattice. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N, N′-dimethylformamide (DMF), N, N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.” In some embodiments, the solvent incorporated into a solvate is of a type or at a level that is physiologically tolerable to an organism to which the solvate is administered (e.g., in a unit dosage form of a pharmaceutical composition).

Piperazine: As used herein, “piperazine” refers to a six membered ring containing two nitrogen atoms at opposite positions in the ring.

Protein of interest: As used herein, the terms “proteins of interest” or “desired proteins” include those provided herein and fragments, mutants, variants, and alterations thereof.

Purine: As used herein, “purine” refers to an aromatic heterocyclic structure, wherein one of the heterocycles is an imidazole ring and one of the heterocycles is a pyrimidine ring.

Pyrimidine: As used herein, “pyrimidine” refers to an aromatic heterocyclic structure similar to benzene, but wherein two of the carbon atoms are replaced by nitrogen atoms.

Pyridopyrimidine: As used herein, “Pyridopyrimidine” refers to an aromatic heterocyclic structure, wherein one of the heterocycles is a purine ring and one of the heterocycles is a pyrimidine ring.

Quinazoline: As used herein, the term, “Quinazoline” refers to an aromatic heterocyclic structure, wherein one of the heterocycles is a benzene ring and one of the heterocycles is a pyrimidine ring.

Stable: As used herein “stable” refers to a compound or entity that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,” “stabilized region” means to make or become stable. In some embodiments, stability is measured relative to an absolute value. In some embodiments, stability is measured relative to a secondary status or state or to a reference compound or entity.

Standard CAR: As used herein, the term “standard CAR” refers to the standard design of a chimeric antigen receptor. The components of a CAR fusion protein including the extracellular scFv fragment, transmembrane domain and one or more intracellular domains are linearly constructed as a single fusion protein.

Stimulus response element (SRE): the term “stimulus response element (SRE), as used herein, is a component of an effector module which is joined, attached, linked to or associated with one or more payloads of the effector module and in some instances, is responsible for the responsive nature of the effector module to one or more stimuli. As used herein, the “responsive” nature of an SRE to a stimulus may be characterized by a covalent or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus. Further, the response of any SRE to a stimulus may be a matter of degree or kind. The response may be a partial response. The response may be a reversible response. The response may ultimately lead to a regulated signal or output. Such output signal may be of a relative nature to the stimulus, e.g., producing a modulatory effect of between 1 and 100 or a factored increase or decrease such as 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more. One non-limiting example of an SRE is a destabilizing domain (DD).

Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

T cell: A T cell is an immune cell that produces T cell receptors (TCRs). T cells can be naïve (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells™ (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naïve T cell and effector memory T cells (T_(EM), decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naïve T cells or TCM). Effector T cells (TE) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for granzyme and perforin as compared to TCM. Other exemplary T cells include regulatory T cells, such as CD4+CD25+(Foxp3+) regulatory T cells and Treg17 cells, as well as Tr1, Th3, CD8+CD28−, and Qa-1 restricted T cells.

T cell receptor: T cell receptor (TCR) refers to an immunoglobulin superfamily member having a variable antigen binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail, which is capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and β chains (also known as TCRα and TCRβ, respectively), or γ and δ chains (also known as TCRγ and TCRδ, respectively). The extracellular portion of TCR chains (e.g., α-chain, β-chain) contains two immunoglobulin domains, a variable domain (e.g., α-chain variable domain or Vα, β-chain variable domain or Vβ) at the N-terminus, and one constant domain (e.g., α-chain constant domain or Cα and β-chain constant domain or Cβ) adjacent to the cell membrane. Similar to immunoglobulin, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs). A TCR is usually associated with the CD3 complex to form a TCR complex. As used herein, the term “TCR complex” refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex can be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRγ chain, and a TCR chain. A “component of a TCR complex,” as used herein, refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains.

Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. Therapeutic agents of the present invention include any of the biocircuit components taught herein either alone or in combination with other therapeutic agents.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is provided in a single dose. In some embodiments, a therapeutically effective amount is administered in a dosage regimen comprising a plurality of doses. Those skilled in the art will appreciate that in some embodiments, a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of such a dosage regimen.

Triazine: As used herein, “triazine” is a class of nitrogen containing heterocycles with a structure similar to benzene, but wherein three carbon atoms are replaced by nitrogen atoms.

Treatment or treating: As used herein, the terms “treatment” or “treating” denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) cancerous cells or other diseased, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.

Tune: As used herein, the term “tune” means to adjust, balance or adapt one thing in response to a stimulus or toward a particular outcome. In one non-limiting example, the SREs and/or DDs of the present invention adjust, balance or adapt the function or structure of compositions to which they are appended, attached or associated with in response to particular stimuli and/or environments.

Variant: As used herein, the term “variant” refers to a first composition (e.g., a first DD or payload), that is related to a second composition (e.g., a second DD or payload, also termed a “parent” molecule). The variant molecule can be derived from, isolated from, based on or homologous to the parent molecule. The term variant can be used to describe either polynucleotides or polypeptides.

VIII. Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention. The present invention is further illustrated by the following nonlimiting examples.

EXAMPLES Example 1. Generation of Novel Ligand Responsive DDs by Mutagenesis Screening Study Design

To engineer constructs that display ligand dependent stability, a candidate ligand binding domain (LBD) is selected and a cell-based screen using yellow fluorescent protein (YFP) as a reporter for protein stability is designed to identify mutants of the candidate LBD possessing the desired characteristics of a destabilizing domain: low protein levels in the absence of a ligand of the LBD, (i.e., low basal stability), large dynamic range, robust and predictable dose-response behavior, and rapid kinetics of degradation (Banaszynski, et al., (2006) Cell; 126(5): 995-1004). The candidate LBD binds to a desired ligand but not endogenous signaling molecules.

The candidate LBD sequence (as a template) is first mutated using a combination of nucleotide analog mutagenesis and error-prone PCR, to generate libraries of mutants based on the template candidate domain sequence. The libraries generated are cloned in-frame at either the 5′- or 3′-ends of the YFP gene, and a retroviral expression system is used to stably transduce the libraries of YFP fusions into NIH3T3 fibroblasts.

The transduced NIH3T3 cells are subjected to three to four rounds of sorting using fluorescence-activated cell sorting (FACS) to screen the libraries of candidate DDs. Transduced NIH3T3 cells are cultured in the absence of the high affinity ligand of the ligand binding domain (LBD), and cells that exhibit low levels of YFP expression are selected through FACS.

Screening Strategy I

The selected cell population is cultured in the presence of the high affinity ligand of the ligand binding domain for a period of time (e.g., 24 hours), at which point cells are sorted again by FACS. Cells that exhibit high levels of YFP expression are selected through FACS and the selected cell population is split into two groups and treated again with the high affinity ligand of the ligand binding domain at different concentrations; one group is treated with the lower concentration of the ligand and the other is treated with a high concentration of the ligand, for a period of time (e.g., 24 hours), at which point cells are sorted again by FACS. Cells expressing mutants that are responsive to lower concentrations of the ligand are isolated.

The isolated cells responsive to the lower concentration of the ligand are treated with the ligand again and cells exhibiting low fluorescence levels are collected 4 hours following removal of the ligand from the media. This fourth sorting is designed to enrich cells that exhibit fast kinetics of degradation (Iwamoto et al., Chem Biol. 2010 Sep. 24; 17(9): 981-988).

Screening Strategy II

The selected cell population is subject to additional one or more sorts by FACS in the absence of high affinity ligand of LBD and cells that exhibit low levels of YFP expression are selected for further analysis. Cells are treated with high affinity ligand of the ligand binding domain, for a period of time (e.g. 24 hours), and sorted again by FACS. Cells expressing high levels of YFP are selected for through FACS. Cells with high expression of YFP are treated with ligand again and cells exhibiting low fluorescence levels are collected 4 hours following removal of the ligand from the media to enrich cells that exhibit fast kinetics of degradation. Any of the sorting steps may be repeated to identify DDs with ligand dependent stability.

The cells are recovered after sorting. The identified candidate cells are harvested and the genomic DNA is extracted. The candidate DDs are amplified by PCR and isolated. The candidate DDs are sequenced and compared to the LBD template to identify the mutations in candidate DDs.

Example 2. Novel DDs Derived from Human PDE5 by Site Directed Mutagenesis

To identify novel destabilizing domain mutations, mutagenic PCR was performed on the open reading frame of human PDE5 catalytic domain (SEQ ID NO. 3) using non-natural nucleotides. The mutant library was ligated in frame with an AcGFP reporter at C-terminus and cloned into pLVX-IRES-Puro vectors. The lentivirus library was then used to infect HEK 293T cells. Cells were selected with puromycin and the library was screened using screening strategies described in Example 1. DNA was extracted from the cell pool, cloned into vectors, and transformed into E. coli. Individual clones were sequenced and cloned in frame with a linker GGSGGGSGG (SEQ ID NO. 77) and AcGFP at C terminus into pLVX.IRES puro. The catalytic domain of wildtype hPDE5 was also cloned into pLVX.IRES. Puro and used as a control. HEK293 cells were transduced with individual clones and selected with puromycin.

HEK293 cells expressing OT-hPDE5N constructs were incubated with 10 μM Sildenafil or vehicle control, DMSO for 48 hours and the stability of hPDE5 mutants was evaluated at the protein level. Cell lysates obtained from hPDE5N construct expressing cells were immunoblotted using the AcGFP antibody (Clonetech, Mountain View, Calif.). Samples were also immunoblotted with the GAPDH antibody to ensure uniform protein loading. The immunoblot demonstrated that OT-hPDE5N-002, OT-hPDE5N-003, OT-hPDE5N-006, OT-hPDE5N-008, OT-hPDE5N-009, OT-hPDE5N-010 showed an increase in PDE5-GFP protein levels with Sildenafil treatment when compared to DMSO treatment suggesting a Sildenafil dependent stabilization of the construct. Further, the hPDE5-GFP levels with DMSO treatment in OT-hPDE5N-002, OT-hPDE5N-003, and OT-hPDE5N-006 was lower than in the wildtype hPDE5 construct, OT-hPDE5N-001, indicating that these constructs are destabilized in the absence of ligand.

The GFP expression in cells expressing hPDE5 constructs was measured by FACS. HEK293 cells expressing OT-hPDE5N-001 to OT-hPDE5N-010 were incubated with 10 μM Sildenafil or vehicle control, DMSO for 48 hours and the mean fluorescence intensity (MFI) was calculated. The stabilization ratio was calculated as the fold change in GFP intensity in Sildenafil treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilizing mutation co-efficient was calculated as the fold change in GFP intensity in the hPDE5 mutant constructs (OT-hPDE5N-002 to OT-hPDE5N-010) compared to the hPDE5 wildtype construct (OT-hPDE5N-001) in the absence of the ligand. Destabilizing mutation co-efficient less than 1 and stabilization ratios greater than 1 are desired in DDs. The results and ratios are presented in Table 19.

TABLE 19 GFP expression in hPDE5 derived DDs Destabilizing MFI Stabilization mutation co- Constructs DMSO Sildenafil ratio efficient OT-hPDE5N-001 11064 25789 2.33 OT-hPDE5N-002 1649 12285 7.45 0.15 OT-hPDE5N-003 1184 7303 6.17 0.11 OT-hPDE5N-004 795 1065 1.34 0.07 OT-hPDE5N-005 6924 29491 4.26 0.63 OT-hPDE5N-006 1542 19211 12.46 0.14 OT-hPDE5N-007 3038 7078 2.33 0.27 OT-hPDE5N-008 1134 13189 11.63 0.10 OT-hPDE5N-009 744 15587 20.95 0.07 OT-hPDE5N-010 5053 21755 4.31 0.46

As shown in Table 19, all constructs demonstrated a stabilization ratio greater than one, indicating that all constructs show ligand (Sildenafil) dependent stabilization. The stabilization ratios of OT-hPDE5N-006, OT-hPDE5N-008, and OT-hPDE5N-009 was greater than 10 indicating strong ligand dependent stabilization. Constructs OT-hPDE5N-002, OT-hPDE5N-003, OT-hPDE5N-005, and OT-hPDE5N-010 showed stabilization ratio in the range of 2 to 10 indicating modest ligand dependent stabilization. The destabilizing mutation coefficient observed with all constructs was less than 1 indicating that all hPDE5 mutants are destabilized as compared to the wildtype hPDE5. Notably, constructs OT-hPDE5N-002, OT-hPDE5N-003, OT-hPDE5N-004, OT-hPDE5N-006, OT-hPDE5N-008, and OT-hPDE5N-009 demonstrated destabilizing mutation coefficients less than 0.2 indicating strong destabilizing in the absence of ligand. OT-hPDE5N-006, OT-hPDE5N-008, and OT-hPDE5N-009 were identified as mutants with desirable characteristics of a DD i.e., low basal expression in the absence of ligand and high expression in the presence of ligand.

Example 3. Sildenafil Dependent Stabilization of hPDE5 Mutants

HEK293 cells expressing OT-hPDE5N-006, OT-hPDE5N-008, or OT-hPDE5N-009 constructs were incubated with varying concentrations of Sildenafil ranging from 0.005 μM to 10 μM, or vehicle control (DMSO) for 48 hours. The stability of hPDE5 mutants was measured using FACS and mean fluorescence intensity (MFI) of GFP was calculated. The stabilization ratio was calculated as the fold change in GFP intensity in Sildenafil treated samples compared to treatment with DMSO (i.e. absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired in DDs. The MFI and stabilization ratios are presented in Table 20.

TABLE 20 Sildenafil Dose titration OT-hPDE5N-006 OT-hPDE5N-008 OT-hPDE5N-009 Sildenafil Stabili- Stabili- Stabili- dose zation zation zation (μM) MFI ratio MFI ratio MFI ratio Untreated 7036 4388 3506 0.0005 6848 0.97 4422 1.01 3513 1.00 0.0015 7239 1.03 4557 1.04 3473 0.99 0.0046 7218 1.03 4397 1.00 3539 1.01 0.0137 7127 1.01 4274 0.97 3683 1.05 0.041 8097 1.15 4395 1.00 4171 1.19 0.123 12248 1.74 4875 1.11 6463 1.84 0.37 22018 3.13 6753 1.54 13076 3.73 1.11 34171 4.86 10495 2.39 23468 6.69 3.33 49220 7.00 17960 4.09 34615 9.87 10 67084 9.53 32480 7.40 47793 13.63

As shown in Table 20, all three constructs showed an increase in stabilization ratio with increasing doses of Sildenafil indicating a ligand dose-dependent stabilization of hPDE5 mutants. The half maximal effective concentration or EC₅₀ was approximately 104.

Sildenafil dependent stabilization of hPDE5 mutants was also measured over a period of time. HEK293 cells expressing OT-hPDE5N-006, OT-hPDE5N-008, and OT-hPDE5N-009 were treated with 10 μM of Sildenafil for 2, 4, 6, 16, 24, and 48 hours. The stability of hPDE5 mutants was measured using FACS and mean fluorescence intensity (MFI) of GFP was calculated. The stabilization ratio was calculated as the fold change in GFP intensity in Sildenafil treated samples compared to treatment with DMSO (i.e. absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired in DDs. The MFI and stabilization ratios are presented in Table 21.

TABLE 21 Time course of Sildenafil treatment OT-hPDE5N-006 OT-hPDE5N-008 OT-hPDE5N-009 Stabili- Stabili- Stabili- Time zation zation zation (h) MFI ratio MFI ratio MFI ratio Parental 675 — 667 — 673 — 0 7921 — 5304 — 4195 — 2 12088 1.54 7290 1.37 6337 1.51 4 14046 1.77 8675 1.64 8159 1.95 6 16710 2.11 10929 2.06 10203 2.43 16 39029 4.93 19034 3.59 26279 6.26 24 51534 6.51 27246 5.14 36516 8.71 48 70811 8.94 34796 6.56 47407 11.30

As shown in Table 21, all three constructs showed an increase in stabilization ratio with increasing duration of treatment with Sildenafil indicating a time-dependent increase in stabilization of hPDE5 mutants.

Example 4. Vardenafil Dependent Stabilization of hPDE5 DDs

Vardenafil has an IC so of 0.7 nM and is a more potent inhibitor of hPDE5 than Sildenafil which has an IC so of 3.9 nM (Doggrell S, et al. (2007) Int J Impot. Res 19(3):281-95). To test if Vardenafil is capable of stabilizing hPDE5 DDs, HEK293 cells expressing OT-hPDE5-006, OT-hPDE5-008, and OT-hPDE5-009 were treated with Vardenafil at concentrations ranging from 0.0005 μM to 10 μM for 48 hours. The stability of hPDE5 mutants was measured using FACS and mean fluorescence intensity (MFI) of GFP was calculated. The stabilization ratio was calculated as the fold change in GFP intensity in Vardenafil treated samples compared to treatment with DMSO (i.e. absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired in DDs. The MFI and stabilization ratios are presented in Table 22.

TABLE 22 Vardenafil Dose Titration OT-hPDE5N-006 OT-hPDE5N-008 OT-hPDE5N-009 Vardenafil Stabili- Stabili- Stabili- dose zation zation zation (μM) MFI ratio MFI ratio MFI ratio Parental 988 — 999 — 980 — 0 11287 — 7971 — 5841 — 0.0005 11770 1.04 7894 0.99 5954 1.02 0.0015 12965 1.15 8099 1.02 6601 1.13 0.0046 18739 1.66 8897 1.12 9946 1.70 0.0137 36940 3.27 12440 1.56 21894 3.75 0.041 58755 5.21 18527 2.32 39580 6.78 0.123 91994 8.15 33614 4.22 63622 10.89 0.37 125140 11.09 61010 7.65 86481 14.81 1.11 144927 12.84 95149 11.94 105930 18.14 3.33 172990 15.33 134306 16.85 123757 21.19 10 188068 16.66 166236 20.86 139165 23.83

As shown in Table 22, all three constructs showed an increase in stabilization ratio with increasing doses of Vardenafil indicating a Vardenafil dose-dependent stabilization of hPDE5 DDs. The half maximal effective concentration or EC₅₀ was approximately 0.1-0.3 μM which was less than the EC₅₀ observed with sildenafil (˜1 μM) indicating that Vardenafil may stabilize hPDE5 DDs more potently than sildenafil.

The ability of Vardenafil to stabilize hPDE5 derived DDs was measured in cell lines stably transduced with hPDE5 DDs e.g. HEK293 cells, HCT-116 cells, and SKOV-3 cells. Cells were transduced with OT-hPDE5N-006, 008, and 009 constructs and incubated with 1 μM or 10 μM Vardenafil or with DMSO for 48 hours. The stability of hPDE5 mutants was measured using FACS and mean fluorescence intensity (MFI) of GFP was calculated. The stabilization ratio was calculated as the fold change in GFP intensity in Vardenafil treated samples compared to treatment with DMSO (i.e. absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired in DDs. The MFI and stabilization ratios are presented in Table 23.

TABLE 23 Vardenafil Dose Titration OT-hPDE5N-006 OT-hPDE5N-008 OT-hPDE5N-009 (hPDE5- F736A) (hPDE5- Y728L) (hPDE5- R732L) Stabili- Stabili- Stabili- zation zation zation Dose MFI ratio MFI ratio MFI ratio DMSO 9604 — 5191 — 3605 — Vardenafil 110603 11.52 68816 13.26 81484 22.60 (1 μM) Vardenafil 143351 14.93 123150 23.72 106403 29.52 (10 μM)

As shown in Table 23, all three constructs showed an increase in stabilization ratio with both doses of Vardenafil indicating a ligand dose-dependent stabilization of hPDE5 DDs. These data are consistent with the results observed with the dose response of Vardenafil observed in Table 22.

Example 5. hPDE5 C Terminus Fusion Proteins

DDs may be positioned upstream or downstream of the payload within an SRE. hPDE5 mutants generated by site mutagenesis as discussed in example 2 are fused at the C-terminus of GFP to test if the hPDE5 mutants can destabilize proteins of interest when fused to the C-terminus of the protein of interest. A linker is placed between GFP and hPDE5 and cloned into pLVX.IRES. Puro. HEK 293T cells stably expressing GFP-hPDE5 (wildtype and mutant) constructs are incubated with 10 μM Sildenafil or 10 μM Vardenafil or DMSO (control) for 48 hours. Following the incubation, mean fluorescence intensity (MFI) is measured using FACS. All hPDE5 (mutant)-GFP constructs are expected to stabilize GFP in the presence of ligand while they are expected to destabilize GFP in the absence of ligand when fused to C terminus.

Example 6. Novel DDs Derived from Human hPDEs by Site Directed Mutagenesis

Known mutations in phosphodiesterases that affect protein stability are identified and utilized to identify novel hPDE derived DDs. Mutations previously identified include, but are not limited to, hPDE5 (I778T), or hPDE6C (H602L), hPDE6C (E790K), hPDE6C(R104W), hPDE6C (Y323N), and hPDE6C (P391L) or hPDE4D (S752A), hPDE4D (S754A), hPDE4D (S752A, S754A), and hPDE4D (E757A, E758A, D759A) (Zhu et al. (2010) Mol Cell Biol. 4379-4390; Alexandre et al. (2015). Endocr. Relat. Cancer 22(4):519-30; Cheguru P. et al. (2015) Mol Cell Neurosci; 64: 1-8; the contents of each of which are incorporated herein by reference in their entirety). Human PDE mutants are fused to a linker, and a reporter gene e.g. GFP. The reporter constructs are transfected into cells such as NIH 3T3 cells and 293T cells. Transfected cells are incubated with appropriate ligand e.g. Sildenafil and Vardenafil for hPDE5 or Apremilast and Roflumilast for hPDE4. Fluorescence signal is measured by FACS and mean fluorescence signal intensity is calculated.

Example 7. Characterization of hPDE5 Mutants Using Thermal Shift Assays

Thermal shift assays can be used to measure the thermal denaturation temperature of a protein as an indicator of its stability in response to different conditions such as pH, ions, salts, additives, drugs, and/or mutations. Additionally, thermal shift assays can be used to understand the correlation between ligand binding, enzymatic activity and stabilization potencies. Human PDE5 mutants are mixed with a thermal assay dye, thermal assay buffer, and ligand (or DMSO control). Samples are also treated with varying concentrations of factors such as drugs, salts, ions, or other parameters. The samples are loaded into an instrument such as a real-time PCR instrument and the temperature ramp rates is set within a range of approximately 0.1-10 degrees Celsius per minute. The fluorescence in each condition is measured at regular intervals, over a temperature range spanning the typical protein unfolding temperatures of 25-95 degrees Celsius.

Example 8. Characterization of hPDE5 Truncation Mutants

The data shown in the previous examples show that the catalytic domain of hPDE5 was a suitable template for the identification of destabilizing domains. Several truncation mutants of the full length hPDE5 were generated to identify regions beyond the catalytic within the catalytic domain that may serve as a template for identifying DDs. The truncation mutants tested included amino acids 535-860 of hPDE5 WT (E535-Q860), amino acids 535 to 836 of hPDE5 WT (E535-S836), amino acids 590 to 860 of hPDE5 WT (M590-Q860), amino acids 590 to 836 of hPDE5 WT (M590-S836), wherein the amino acids positions are with respect to SEQ ID NO. 1. The DD mutation R732L was incorporated into all truncation mutants and its destabilizing potential was compared to E535-Q860 wildtype by fusing the mutants to an SG linker and reporter, AcGFP. HCT-116 cells were stably transduced with the constructs described above and treated with 10 μM stabilizing ligands, Sildenafil, or Vardenafil or DMSO for 48 hours. As a control, parental untransduced cells and cells expressing OT-hPDE5N-001, or OT-hPDE5N-028 were also included in the analysis. The mean fluorescence intensity (MFI) of GFP was analyzed by FACS. The stabilization ratio was calculated as the fold change in GFP intensity in Sildenafil treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilizing mutation coefficient was calculated as the fold change in GFP intensity in the hPDE5 mutant constructs compared to the hPDE5 wildtype construct (OT-hPDE5N-019) in the absence of the ligand. Destabilizing mutation coefficients less than 1 are desirable. Stabilization ratios greater than 1 are desired in DDs. The results and ratios are presented in Table 24.

TABLE 24 Expression of hPDE5 truncation mutants Sildenafil Vardenafil Destabilizing MFI Stabilization Stabilization mutation Construct DMSO Sildenafil Vardenafil ratio ratio coefficient Parental 2093 3735 3200 1.78 1.53 — OT-hPDE5N-001 42973 53736 49344 1.25 1.15 — OT-hPDE5N-028 150356 187845 170129 1.25 1.13 — OT-hPDE5N-019 64942 66860 67388 1.03 1.04 — OT-hPDE5N-020 9248 55636 93216 6.02 10.08 0.14 OT-hPDE5N-021 4542 4608 5188 1.01 1.14 0.07 OT-hPDE5N-022 3395 3434 3523 1.01 1.04 0.05 OT-hPDE5N-023 2689 2686 2660 1.00 0.99 0.04 OT-hPDE5N-024 2809 2663 2770 0.95 0.99 0.04

As shown in Table 24, OT-hPDE5N-020 construct consisting of E535-Q860 amino acids of hPDE5 with the R732L mutant displayed the strongest ligand dependent stabilization with both ligands with the highest stabilization ratio. The destabilizing mutation coefficient was also calculated for constructs 020-024 and this analysis showed that the constructs were all effective in destabilizing the payload. This suggests that the removal of residues from the catalytic domain does not appear to enhance the ligand dependent stabilization potential of hPDE5.

Protein expression analysis of the truncation mutants was performed in parallel via western blot. HCT 116 cells expressing OT-hPDE5N-019 to OT-hPDE5N-024 were treated with 10 μM Vardenafil for 24 hours and immunoblotted for AcGFP using anti AcGFP antibodies (catalog no. 63277, Clonetech, Mountain View, Calif.). GAPDH levels were also analyzed to ensure even protein loading. OT-hPDE5N-020 construct consisting of E535-Q860 i.e. amino acids 535 to 860 of hPDE5 WT (SEQ ID NO. 1) with the R732L mutant showed vardenafil dependent stabilization. This result is consistent with the FACS analysis. Stabilization was not observed with OT-hPDE5N-021, while constructs OT-hPDE5N-022, OT-hPDE5N-023, and OT-hPDE5N-024 showed no AcGFP expression, both in the presence and absence of vardenafil, indicating that the expression of the construct in these mutants is below the detection levels of the western blot method.

Example 9. Characterization of hPDE5 Combination Mutants

Single destabilizing mutants identified were combined to test if the combining two or more mutations generates domains with greater destabilizing potential either additively or synergistically. Desirable qualities of a DD include, low expression of the SRE in the absence of ligand and ligand dependent stabilization of the SRE. Constructs were generated using DDs linked to GFP using an SG linker. HCT-116 cells were stably transduced with the constructs described above and treated with 10 μM stabilizing ligands, Sildenafil, or Vardenafil or DMSO for 48 hours. As a control, parental untransduced cells and cells expressing OT-hPDE5N-001, or OT-hPDE5N-028 were also included in the analysis. The mean fluorescence intensity (MFI) of GFP was analyzed by FACS. The stabilization ratio was calculated as the fold change in GFP intensity in Sildenafil treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilizing mutation coefficient was calculated as the fold change in GFP intensity in the hPDE5 mutant constructs compared to the hPDE5 wildtype construct (OT-hPDE5N-019) in the absence of the ligand. Destabilizing mutation coefficient less than 1 are desirable. Stabilization ratios greater than 1 are desired in DDs. The results and ratios are presented in Table 25.

TABLE 25 Expression of hPDE5 combination mutants Sildenafil Vardenafil Destabilizing MFI Stabilization Stabilization mutation Construct DMSO Sildenafil Vardenafil ratio ratio coefficient Parental 2093 3735 3200 1.78 1.53 — OT-hPDE5N-001 42973 53736 49344 1.25 1.15 — OT-hPDE5N-028 150356 187845 170129 1.25 1.13 — OT-hPDE5N-019 64942 66860 67388 1.03 1.04 — OT-hPDE5N-025 12633 13862 53848 1.10 4.26 0.19 OT-hPDE5N-026 8625 48592 109667 5.63 12.72 0.13

As shown in Table 25, only the OT-hPDE5N-026 construct with R732L and D764N mutations showed Sildenafil dependent stabilization with a ratio of 5.63. Similar results were obtained with OT-hPDE5N-026 upon treatment with Vardenafil. OT-hPDE5N-025 however only showed Vardenafil dependent stabilization with a ratio of 4.26. Both constructs were destabilized in the absence of ligand. As expected, hPDE5 wildtype constructs did not show any significant ligand dependent stabilization. These data suggest that the strategy of combining mutations may be used to identify improved DDs as well as DDs that are stabilized by specific ligands.

Protein expression analysis of the combination mutants was performed in parallel via western blot. HCT 116 cells expressing OT-hPDE5N-025, and 26 were treated with 10 μM Vardenafil for 24 hours and immunoblotted for AcGFP using anti AcGFP antibodies (catalog no. 63277, Clonetech, Mountain View, Calif.). GAPDH levels were also analyzed to ensure even protein loading. OT-hPDE5N-025 with hPDE5 (F736A, D764N) and OT-hPDE5N-026 with hPDE5 (R732L, D764N) constructs consisting showed Vardenafil dependent stabilization of AcGFP protein levels as measured via western blotting. This result is consistent with the FACS analysis.

The response of hPDE5 combination mutants to increasing doses of sildenafil was tested. HCT116 cells transduced with hPDE5 constructs were treated with Sildenafil for 24 hours at doses ranging from 0.04 μM to 30 μM. Mean fluorescence intensity (MFI) was measured by FACS. Parental HCT116 and cells expressing OT-hPDE5N-019 wildtype construct were also included as controls. The response of the combination mutants to Sildenafil was compared to the response of the single mutant construct OT-hPDE5N-020. The stabilization ratio indicated as SR in Table 26 was calculated as the fold change in GFP intensity in Sildenafil treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilizing mutation coefficient ratio was calculated as the fold change in GFP intensity in the hPDE5 mutant constructs compared to the hPDE5 wildtype construct (OT-hPDE5N-019) in the absence of the ligand. Destabilizing mutation coefficient ratios less than 1 are desirable. Stabilization ratios greater than 1 are desired in DDs. The results and stabilization ratios are presented in Table 26.

TABLE 26 Sildenafil dose response of combination mutants Dose (μM OT-hPDE5N-019 OT-hPDE5N-020 OT-hPDE5N-025 OT-hPDE5N-026 Sildenafil) MFI SR MFI SR MFI SR MFI SR DMSO 62116 — 6172 — 10736 — 7093 — 30 75478 1.22 47992 7.78 26837 2.50 69864 9.85 10 — — 27693 4.49 13263 1.24 34587 4.88 3.33 — — 23549 3.82 10384 0.97 22392 3.16 1.11 — — 15952 2.58 9636 0.90 10353 1.46 0.37 — — 12739 2.06 9802 0.91 6961 0.98 0.12 — — 6441 1.04 8619 0.80 6603 0.93 0.04 — — 5299 0.86 8688 0.81 5948 0.84

As shown in Table 26, the stabilization ratios for OT-hPDE5N-026 construct obtained across multiple doses of sildenafil were higher than the single mutant OT-hPDE5N-020 construct. The combination mutant 025, achieved stabilization ratios much lower than the 026 construct for any given ligand dose. The destabilizing mutation co-efficient ratios obtained for the OT-hPDE5N-020 (ratio=0.1), OT-hPDE5N-025 (ratio=0.17), and OT-hPDE5N-026 (ratio =0.1) indicate that all constructs are destabilized in the absence of the ligand. These data indicate that the combination mutant OT-hPDE5N-026 is strong DD candidate when the stabilizing ligand is Sildenafil.

The response of hPDE5 combination mutants to increasing doses of Vardenafil was tested. HCT116 cells transduced with hPDE5 constructs were treated with Vardenafil for 24 hours at doses ranging from 0.04 μM to 30 μM. Mean fluorescence intensity (MFI) was measured by FACS. Parental HCT116 and cells expressing OT-hPDE5N-019 comprising hPDE5 wildtype construct were also included as controls. The response of the combination mutants to Vardenafil was compared to the response of the single mutant construct OT-hPDE5N-020. The stabilization ratio (SR) was calculated as the fold change in GFP intensity in Vardenafil treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilizing mutation coefficient ratio was calculated as the fold change in GFP intensity in the hPDE5 mutant constructs compared to the hPDE5 wildtype construct (OT-hPDE5N-019) in the absence of the ligand. Destabilizing mutation co-efficient ratios less than 1 are desirable. Stabilization ratios greater than 1 are desired in DDs. The results and stabilization ratios are presented in Table 27.

TABLE 27 Vardenafil dose response of combination mutants Dose (μM OT-hPDE5N-019 OT-hPDE5N-020 OT-hPDE5N-025 OT-hPDE5N-026 Vardenafil) MFI SR MFI SR MFI SR MFI SE DMSO 62116 — 7829 — 7694 — 5156 30 70898 1.14 75323 9.62 46109 5.99 55843 10.83 10 — — 39089 4.99 29971 3.90 35191 6.83 3.33 — — 35564 4.54 29520 3.84 40408 7.84 1.11 — — 30879 3.94 12634 1.64 32868 6.37 0.37 — — 24847 3.17 6667 0.87 14543 2.82 0.12 — — 16866 2.15 7314 0.95 10676 2.07 0.04 — — 14745 1.88 6174 0.80 5497 1.07

As shown in Table 27, the stabilization ratios for OT-hPDE5N-026 construct obtained across multiple doses of Vardenafil were higher than the single mutant OT-hPDE5N-020 construct. The combination mutant 025, achieved stabilization ratios much lower than the 026 construct for any given ligand dose. The destabilizing mutation coefficient obtained for the OT-hPDE5N-020 (ratio=0.13), OT-hPDE5N-025 (ratio=0.12), and OT-hPDE5N-026 (ratio=0.08) indicate that all constructs are destabilized in the absence of the ligand. These data indicate that the combination mutant OT-hPDE5N-026 is strong DD candidate when the stabilizing ligand is Vardenafil.

The response of hPDE5 combination mutants to increasing doses of Tadalafil was tested. HCT116 cells transduced with hPDE5 constructs were treated with Tadalafil for 24 hours at doses ranging from 0.14 to 100 μM. Mean fluorescence intensity (MFI) was measured by FACS. Parental HCT116 and cells expressing OT-hPDE5N-019 comprising hPDE5 wildtype construct were also included as controls. The response of the combination mutants to Tadalafil was compared to the response of the single mutant construct OT-hPDE5N-020. The stabilization ratio (SR) was calculated as the fold change in GFP intensity in Tadalafil treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilizing mutation coefficient was calculated as the fold change in GFP intensity in the hPDE5 mutant constructs compared to the hPDE5 wildtype construct (OT-hPDE5N-0019) in the absence of the ligand. Destabilizing mutation coefficient less than 1 are desirable. Stabilization ratios greater than 1 are desired in DDs. The results and stabilization ratios are presented in Table 28.

TABLE 28 Tadalafil dose response of combination mutants Dose (μM OT-hPDE5N-019 OT-hPDE5N-020 OT-hPDE5N-025 OT-hPDE5N-026 Vardenafil) MFI SR MFI SR MFI SR MFI SR DMSO 36453 — 3806 — 6043 — 3746 — 100 58468 1.60 34041 8.94 23542 3.90 30029 8.02 33.33 — — 44713 11.75 20464 3.39 30028 8.02 11.11 — — 30641 8.05 13789 2.28 24499 6.54 3.7 — — 26480 6.96 9515 1.57 17317 4.62 1.23 — — 20458 5.38 8634 1.43 9283 2.48 0.41 — — 13206 3.47 6141 1.02 6044 1.61 0.14 — — 8512 2.24 5768 0.95 4682 1.25

As shown in Table 28, the stabilization ratios for OT-hPDE5N-026 construct obtained across multiple doses of Tadalafil were higher than the single mutant OT-hPDE5N-020 construct, except at the 100 μM concentration of the drug. The combination mutant OT-hPDE5-025, achieved stabilization ratios much lower than the OT-hPDE5-026 construct for any given ligand dose. The destabilizing mutation coefficient obtained for the OT-hPDE5N-020 (ratio=0.1), OT-hPDE5N-025 (ratio=0.17), and OT-hPDE5N-026 (ratio=0.1) indicate that all constructs are destabilized in the absence of the ligand. These data indicate that the combination mutant OT-hPDE5N-026 is strong DD candidate when the stabilizing ligand is Tadalafil.

The stabilization ratios obtained for the highest concentrations of all three ligands, Sildenafil, Vardenafil and Tadalafil for each of the constructs were compared and are shown Table 29.

TABLE 29 Comparative analysis of hPDE5 ligands Stabilization ratio OT- OT- OT- OT- hPDE5N- hPDE5N- hPDE5N- hPDE5N- Ligand 019 020 025 026 Sildenafil 1.22 7.78 2.5 9.85 (30 μM) Vardenafil 1.14 9.62 5.99 10.83 (30 μM) Tadalafil 1.60 8.94 3.9 8.02 (100 μM)

As shown in Table 29, based on the stabilization ratios it was evident that OT-hPDE5N-026 was effectively stabilized by all three ligands, in comparison to both the single mutant construct as well as the other combination mutant construct (OT-hPDE5N-025). Both combination mutants were most effectively stabilized by Vardenafil, which is the most potent inhibitor of hPDE5. Construct OT-hPDE5N-025 was stabilized more effectively by Tadalafil than Sildenafil, although only by a small margin-this observation is noteworthy as Sildenafil is a much stronger inhibitor of hPDE5 than Tadalafil

The response of hPDE5 combination mutants to increasing duration treatment with sildenafil was tested. HCT116 cells transduced with hPDE5 constructs were treated with Sildenafil at 0.5 or 5 μM for 0-72 hours. Mean fluorescence intensity (MFI) was measured by FACS. The response of the combination mutants to Sildenafil was compared to the response of the single mutant construct OT-hPDE5-020. The stabilization ratio was calculated as the fold change in GFP intensity in Sildenafil treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilizing mutation coefficient was calculated as the fold change in GFP intensity in the hPDE5 mutant constructs compared to the hPDE5 wildtype construct (OT-hPDE5N-019) in the absence of the ligand. Destabilizing mutation coefficient less than 1 are desirable. Stabilization ratios greater than 1 are desired in DDs. The results and stabilization ratios for time course experiments with 5 or 0.5 μM are presented in Table 30 and Table 31 respectively.

TABLE 30 Response to Sildenafil (5 μM) over time OT-hPDE5N-020 OT-hPDE5N-025 OT-hPDE5N-026 Stabili- Stabili- Stabili- Time zation zation zation (hours) MFI ratio MFI ratio MFI ratio 0 2668 — 3364 — 2769 — 72 58479 21.92 26995 8.02 90413 32.65 48 46604 17.47 22061 6.56 65233 23.56 24 20955 7.85 16705 4.97 27962 10.10 20 15529 5.82 13280 3.95 21603 7.80 16 14855 5.57 12280 3.65 17600 6.36 12 9097 3.41 9199 2.73 10451 3.77 8 5672 2.13 7021 2.09 6179 2.23 4 4236 1.59 6314 1.88 4266 1.54

TABLE 31 Response to Sildenafil (0.5 μM) over time OT-hPDE5N-020 OT-hPDE5N-025 OT-hPDE5N-026 Stabili- Stabili- Stabili- Time zation zation zation (hours) MFI ratio MFI ratio MFI ratio 0 2727 — 3245 — 2611 — 72 58786 21.56 6906 2.13 31835 12.19 48 45754 16.78 6682 2.06 27231 10.43 24 20524 7.53 6144 1.89 17848 6.84 20 16142 5.92 6003 1.85 13318 5.10 16 14469 5.31 5600 1.73 11390 4.36 12 9058 3.32 5122 1.58 8126 3.11 8 5461 2.00 5083 1.57 5742 2.20 4 4127 1.51 5322 1.64 3870 1.48

As shown in Table 30 and Table 31, the stabilization ratios for OT-hPDE5N-026 construct obtained over time at 5 μM dose of Sildenafil were higher than the single mutant OT-hPDE5N-020 construct. However, this pattern was reversed at lower dose of 0.5 μM where the single mutant construct showed greater stabilization. The combination mutant OT-hPDE5-025, achieved stabilization ratios much lower than the OT-hPDE5-026 construct for both ligand doses at any given time. These data indicate that the choice of a suitable DD may sometime depend on the duration of the ligand treatment desired as well as the concentration of the ligand that is available for use in a system.

Example 10. Mutagenesis of DD Hotspots

The analysis of mutants identified by site directed mutagenesis identified amino acid hotspots whose mutation confers destabilization and ligand dependent stabilization properties to hPDE5. To improve the DD characteristics of these constructs, the amino acid at the hotspot position is mutated to any of the known amino acids, including, but not limited to lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan, alanine, isoleucine, leucine, phenylalanine, valine, proline, and glycine. The library of hotspot mutations is generated by site directed mutagenesis and each of the mutants in the library is fused to a reporter protein e.g. AcGFP via a linker. The properties of the DDs are analyzed in the presence and absence of ligands via western blot and FACS as previously described. Ligands that are evaluated include, but not limited Sildenafil, Vardenafil and Tadalafil.

Example 11. Optimizing Biocircuit Behavior

The biocircuits of the invention comprises multiple modules which can be optimized. Libraries of each of the components is generated to allow for the rapid generation of new constructs with desired behaviors. Ligand pharmacokinetics is a powerful tool for payload specific tuning in vivo, which can be used to shift the ligand response curve of the effector module to the left or right depending on the modulating factors. Several modulating factors are tested, including, but not limited to the ligand dose, concentrations, magnitude, duration, and route of administration. Destabilizing domains can also be modified to improve biocircuit behavior. The destabilizing domain is the core determinant of the dynamic range of the biocircuit. Depending on the DD selected, the ligand response curve of the effector module can be shifted up or down. The nature, position of the DD within the effector module as well as the number of DDs within an effector module are modified. DD selection is also altered depending on its degradation kinetics desired. Promoters that transcriptionally control the expression of the SREs are optimized. Choice of promoter impacts the basal-off state and affects the dynamic range of stabilization. Further, promoter choice contributes to the extent of stabilized payload produced. Other optimizable elements of the biocircuits include vector, translational elements, leader sequence, placement of the components within the SRE, codon selection, protease sites, linkers, and mRNA stability.

Example 12. Vardenafil Dose Response Curve in hPDE5 Mutants

Combination mutants were generated as described in Example 9. The response of hPDE5 combination mutants to increasing doses of Vardenafil was tested in HCT116 cells transduced with hPDE5 constructs were treated with Vardenafil for 48 hours at doses ranging from 0.1 to 10 μM or vehicle control (DMSO). Mean fluorescence intensity (MFI) of GFP was measured by FACS. The response of the combination mutants to Vardenafil was compared to the response of the single mutant construct OT-hPDE5N-020. The fold change in MFI over DMSO is presented in Table 32.

TABLE 32 Vardenafil dose response Sildenafil (μM) OT-hPDE5-020 OT-hPDE5-025 OT-hPDE5-026 0.01 4026 4444 3815 0.04 6970 4934 3839 0.12 14832 5133 4870 0.37 26429 5007 8107 1.11 34932 5751 21857 3.33 49739 10516 49105 10.00 51306 24927 64734

As shown in Table 32, the ligand dependent stabilization obtained for OT-hPDE5N− 020 construct was observed at lower doses i.e. 0.1 and 0.3 micro molar concentrations of Vardenafil compared to the other two constructs, indicating that very low doses of the ligand are sufficient to stabilize this hPDE5 mutant. The OT-hPDE5N-026 construct did not show ligand dependent stabilization at similar doses. However, at the highest dose of Vardenafil the GFP expression obtained was much higher than OT-hPDE5N-020. In contrast, OT-hPDE5N-025 showed very little expression of GFP at lower doses and only modest GFP expression compared to the other two constructs at the higher doses. These experiments indicate that combination of single hPDE5 DD mutations produces synergistic effects not predicted by the properties of the single mutants. Thus, based on the properties of the payload of interest and the expression levels desired, suitable hPDE5 DDs may be selected.

Example 13. DD Regulated Immunotherapeutic Agent Expression In Vitro

Immunotherapeutic payloads of the invention such as cytokines (e.g. IL2, IL12, IL15, IL15-IL15Rα), chimeric antigen receptors (CD19 CAR), regulatory proteins (e.g. FOXP3), safety switch (e.g. Caspase 9) are fused to any of the DDs described herein and cloned into expression vectors. Optional linkers and cleavage sites are added to optimize the confirmation of the effector module.

To test ligand dependent immunotherapeutic payload production, cells are plated in a growth media e.g. DMEM with 10% FBS and incubated overnight at 37° C., 5% CO2. Cells are transiently transfected or transduced with the constructs using Lipofectamine 2000 and incubated for 48 hrs. Following the incubation, growth media is exchanged for media containing ligands (e.g. Sildenafil, Vardenafil, and Tadalafil). Following 24-hour incubation with ligand, cells are lysed and immunoblotted using antibodies specific to the payload. For secreted payloads, such as cytokines, the growth media is harvested from the cultures expressing the effector modules of the present invention. Similar to the immunoblot, the media is assayed for the expression of the payload using methods known in the art such as ELISA and MSD assay. For cell surface expressed payloads, the expression of the payload is also assayed using methods of cell surface expression analysis that are known in the art such as flow cytometry. Expression of the payload obtained in the presence of ligand is compared to expression in the absence of ligand. Increase in the levels of the payload with increase in ligand concentrations and/or duration of treatment is indicative of DD mediated regulation of the payload. Expression is also compared to parental untransduced cells as well as cells expressing immunotherapeutic payloads that are not appended to DD (i.e. expressed constitutively).

Example 14. DD Regulated Immunotherapeutic Agent Function In Vitro

To test if DD regulated IL12's expression is capable of activating IL12 signaling. Human T-cells are isolated from PBMCs and activated with phytohemagglutinin (PHA, 2 μg/ml) for 3 days, followed by treatment with 50 IU/ml of Interleukin-2 (IL2) for 24 hrs. Cells are washed, resuspended in fresh media and rested for 4 hrs. T cells are transduced with DD-regulated IL12 constructs and treated with the ligand (e.g. Sildenafil, Vardenafil, and Tadalafil) based on the DD utilized or the vehicle control. Activation via IL12 results in STAT4 phosphorylation. Additionally, IL12 promotes the differentiation of naïve T cells into Th1 cells, which results in the secretion of IFN gamma from T cells. Cells are harvested and STAT4 phosphorylation is measured using STAT4 antibody (Cell Signaling Technology, Danvers, Mass.). Cell supernatants and cell lysates are analyzed for IFNgamma. In the presence of ligand, cells expressing DD regulated IL12 are expected to have increased STAT4 phosphorylation and increased expression of IFNgamma.

IL15 and IL15/IL15Ra fusion molecule can confer the memory phenotype on T cells and increase proliferation of NK cells (Hurton L et al. (2016), PNAS, 113: E7788-7797). This dependence of NK cell proliferation on cytokines can be used to test the functionality of DD regulated or constitutively expressed cytokines and cytokine fusion proteins. NK-92 cell activation in response to ligand treatment is evaluated by FACS using a panel of markers whose increased expression is associated with NK activation. These include NKG2D, CD71, CD69; chemokine receptors such as CCR5, CXCR4, and CXCR3, Perforin, Granzyme B and Interferon gamma (IFNg). Expression of DD regulated IL15 or IL15-IL15Ra fusion molecules is expected to increase NK cell activation. To evaluate the effect by ligand-dependent stabilization of IL15 and IL15-IL15Ra on primary T cells, T cells are transduced with DD-IL15Ra constructs. T cell proliferation and memory phenotype markers (e.g. CD62L) are measured either in the presence or absence of ligand by using flow cytometry.

To test the ability of DD regulated CD19 CAR cells to kill target cells in vitro, primary T cell populations are transduced with DD regulated CD19 CAR constructs are co cultured with K562 cells expressing CD19 (target cells) in the presence or absence of the ligand specific to the DD. Multiple combinations of T cells and target cells are set up. These include DD regulated CAR expressing T cells co cultured with K562 cells (in the presence or absence of the ligand), T cells co cultured with K562 cells expressing CD19 and K562 cells expressing CD19 without T cell co culture. Additional controls include target cells only; untransduced T cells; T cells transduced with empty vector. Target cells are treated with Mitomycin C to prevent their proliferation. The K562 cells are fluorescently labelled with NucLight Red and co cultured with T cells for 300 hours. Cell death is monitored by labelling cells with Annexin V and the cell death in target K562 cells is monitored by evaluating cells that are positive for both Annexin V and NucLight Red using the IncuCyte® Live Cell Analysis System (Essen Biosciences, Ann Arbor, Mich.). Target cell killing is expected with the DD regulated CAR constructs only in the presence of ligand and when K562 target cells ectopically expressing CD19 are utilized. No cell killing is expected in untreated controls of the same co-culture set up and when T cells are co cultured with parental K562 cells that do not express CD19 in the presence or absence of ligand. Constitutive constructs are predicted to show cell killing both in the presence of ligand.

Example 15. DD Regulated Immunotherapeutic Agent Function In Vivo

The ligand-dependent in vivo function of DD-regulated immunotherapeutic payloads is characterized by evaluating the ability of T cells expressing DD regulated constructs to inhibit the growth of established tumors upon treatment with the ligands of the invention. Tumor cells such as HCT116 cells are subcutaneously xenografted into mice. T cells stably transduced with a DD regulated constructs described herein are injected intravenously into mice. Approximately, two weeks after injection when the tumors reach a size of approximately 300 cubic mm, mice are dosed with the ligand (e.g. Sildenafil, Vardenafil, and Tadalafil) or vehicle control at varying concentrations every two days. Tumor volume and body weight are monitored twice a week. Plasma and tumor samples are also collected after the last dosing of the ligand and the payload levels as well as the ligand levels are measured. Tumor volume is expected to be significantly smaller in mice treated the ligand compared to vehicle treated animals. Tumor payload levels are expected to positively correlate with tumor volume.

To measure if compositions of the invention promote immune cell persistence, T cells transduced with DD-IL15/IL15Ra constructs are injected into mice. Mice are treated with the ligand (e.g. Sildenafil, Vardenafil, and Tadalafil) and monitored over a period of 40-50 days. T cells transduced with a constitutive OT-IL15-008 construct and untransduced parental T cells are injected into separate mice as controls. Blood is routinely withdrawn from the mice and tested for the presence of T cells. Mice treated with the ligand are expected to retain T cells expressing hPDE5 DD-IL15-IL15Ra while T cells in vehicle treated mice are not expected to persist.

Example 16. Co-Expression of DD Regulated Payloads

Toxicity related to systemic administration of interleukins can be circumvented by using CAR-T cells to deliver interleukins to the target tissue. This combinatorial approach also has greater anti-tumor activity than interleukin and CAR therapy alone. Cells are co-transfected with CD19 CAR (constitutive or DD regulated) and DD-Interleukin e.g. DD-IL2, DD-IL12, DD-IL15 and DD-IL15/IL15Ra constructs. Transfected cells are treated with stabilizing ligands depending on the DD utilized. CD19 CAR expression is evaluated by immunoblotting for CD3 zeta. DD-IL2, DD-IL12, DD-IL15 and DD-IL15/IL15Ra expression in the media is measured by ELISA.

A DD-regulated caspase 9 has the potential improve safety and minimize the toxicity associated with CD19 CAR therapy. Cells are co-transfected with CD19 CAR (constitutive or DD regulated) and DD-caspase 9 constructs. Transfected cells are treated with stabilizing ligands depending on the DD utilized. CD19 CAR and caspase 9 expression are evaluated by immunoblotting for CD3 zeta and caspase 9 respectively.

Example 17. Sildenafil and Tadalafil Dose Response Curve in hPDE5 Mutants

Combination mutations were generated as previously described in Example 9. The response of HCT 116 cells expressing hPDE5 combination mutants to increasing doses ranging from 0.1 μM to 100 μM of Tadalafil and Sildenafil for 48 hours or vehicle control (DMSO) was tested. Mean fluorescence intensity (MFI) was measured by FACS. The response of the combination mutants to ligand treatment was compared to the single mutant construct, OT-hPDE5N-020. The MFI with ligand treatment is shown in Table 33 and Table 34.

TABLE 33 Tadalafil dose response Tadalafil (μM) OT-hPDE5-020 OT-hPDE5-025 OT-hPDE5-026 0.14 3616 4328 3720 0.41 5811 5002 3804 1.23 11694 5503 4810 3.70 24185 5744 7893 11.11 40380 8299 24617 33.33 57233 15831 52927 100.00 72587 31020 84334

TABLE 34 Sildenafil dose response Sildenafil (μM) OT-hPDE5-020 OT-hPDE5-025 OT-hPDE5-026 0.14 3771 4398 3401 0.41 5463 4832 3681 1.23 10479 5031 4477 3.70 22441 4974 7625 11.11 35561 5675 24104 33.33 52029 9677 51416 100.00 101226 37494 136010

As shown in Table 33 and Table 34, ligand dose dependent stabilization was obtained with all three constructs. The dose dependent stabilization at lower doses (e.g., up to 11 μM of Sildenafil and Tadalafil) is more evident in the single mutant construct, OT-hPDE5-020, compared to the other two constructs. At higher concentrations of ligand, both OT-hPDE5-025 and OT-hPDE5-026 showed ligand dependent stabilization. In fact, OT-hPDE5-026 construct demonstrated much higher MFI values at 33 and 100 compared to both OT-hPDE5-020 and OT-hPDE5-025. These experiments indicate that combination of single hPDE5 DD mutations produces synergistic effects not predicted by the properties of the single mutants. Thus, suitable hPDE5 DDs may be selected based on the properties of the payload of interest and the expression levels desired.

Example 18. Kinetics of hPDE5 Mutants

The off kinetics of hPDE5 mutants, OT-hPDE5-020 and OT-hPDE5-026 was tested by treating HCT 116 cells expressing these constructs or the wild type control construct OT-hPDE5-019. Cells were treated with ligand for 48 hours. The media containing the ligand was removed and fresh media without the ligand was added. MFI was analyzed by FACS at 0 hours (i.e., prior to the beginning of the experiment), 48 hours after ligand treatment, as well as at several time points after the ligand washout, up until 96 hours. The stabilization ratio was calculated as the fold change in GFP intensity in ligand treated samples compared to the GFP intensity in the absence of ligand with the same construct. The results are shown in Table 35.

TABLE 35 Off-Kinetics OT-hPDE5-020 OT-hPDE5-026 OT-hPDE5-019 (WT) Stabili- Stabili- Stabili- Time zation zation zation (hrs) MFI Ratio MFI Ratio MFI Ratio 0 3675 — 3648 — 45512 — 48 71538 19.47 57105 15.65 102137 2.24 50 63832 17.37 41074 11.26 97225 2.14 52 51544 14.03 26926 7.38 96264 2.12 54 41800 11.37 16391 4.49 95410 2.10 56 25521 6.94 7700 2.11 81733 1.80 60 19598 5.33 5635 1.54 84323 1.85 72 8062 2.19 4062 1.11 70151 1.54 80 6427 1.75 3587 0.98 63208 1.39 96 4319 1.18 3443 0.94 55918 1.23

As shown in Table 35, the stabilization ratio of hPDE5 mutants decreased following removal of the ligand. The stabilization ratio values decreased more quickly in the combination mutant OT-hPDE5-026 compared to OT-hPDE5-020 construct suggesting the destabilization of the DD is achieved more quickly in the OT-hPDE5-026 construct. The ligand dependent stabilization as indicated by the stabilization ratio, achieved with OT-hPDE5-026 construct is lower than OT-hPDE5-020 construct, which may also contribute to the superior off kinetics seen with the OT-hPDE5-026 construct.

Example 19. Behavior of hPDE5 C Terminal Mutants

The ability of hPDE5 mutants to tune the expression of payloads when appended to the C terminal of the payload was tested. OT-hPDE5C-030 was compared to OT-hPDE5C-036. NIH 3T3 cells were incubated with varying doses of Sildenafil or Vardenafil for 48 hours and MFIs were analyzed by FACS. The stabilization ratio (SR) was calculated as the fold change in GFP intensity in ligand treated samples compared to the GFP intensity in the absence of ligand with the same construct. The results are shown in Table 36.

TABLE 36 hPDE5 C-terminal mutant's response to Sildenafil and Vardenafil Ligand OT-hPDE5C-030 OT-hPDE5C-036 concentration Sildenafil Vardenafil Sildenafil Vardenafil (nM) MFI SR MFI SR MFI SR MFI SR 0 102 — 104 — 1476 — 1410 — 100 278 2.73 971 9.34 1639 1.11 1614 1.14 300 562 5.51 1165 11.20 1648 1.12 1656 1.03 1000 1039 10.19 1235 11.88 1780 1.21 1686 1.02 3000 1276 12.51 1299 12.49 1784 1.21 1721 1.02 10000 1442 14.14 1309 12.59 1888 1.28 1721 1.00

As shown in Table 36, OT-hPDE5C-030 construct showed stabilization ratios greater than one, indicating that both Vardenafil and Tadalafil ligand could stabilize GFP expression. As expected, the wildtype construct, OT-hPDE5C-036 did not stabilization much greater than one, indicating that virtually no stabilization was achieved with the wildtype construct.

GFP expression was also measured at 24 and 48 hours to monitor the dependence of hPDE5-DD GFP constructs on the duration of ligand exposure. The results are shown in Table 37 and Table 38A, where SR indicates stabilization ratio.

TABLE 37 Dose response of hPDE5 C-terminal mutants with Sildenafil treatment Sildenafil OT-hPDE5C-030 OT-hPDE5C-036 concentration 24 hours 48 hours 24 hours 48 hours (nM) MFI SR MFI SR MFI SR MFI SR 0 108 102 — 1385 — 1476 — 100 243 2.25 278 2.73 1495 1.08 1639 1.11 300 380 3.52 562 5.51 1565 1.13 1648 1.12 1000 575 5.32 1039 10.19 1669 1.21 1780 1.21 3000 666 6.17 1276 12.51 1831 1.32 1784 1.21 10000 — — 1442 14.14 — — 1888 1.28

TABLE 38A Dose response of hPDE5 C-terminal mutants with Vardenafil treatment Vardenafil OT-hPDE5C-030 OT-hPDE5C-036 concentration 24 hours 48 hours 24 hours 48 hours (nM) MFI SR MFI SR MFI SR MFI SR 0 108 104 — 1360 — 1410 — 100 684 6.33 971 9.34 1439 1.06 1614 1.14 300 761 7.05 1165 11.20 1730 1.27 1656 1.17 1000 863 7.99 1235 11.88 1721 1.27 1666 1.18 3000 856 7.93 1299 12.49 1606 1.18 1721 1.22 10000 889 8.23 1309 12.59 1678 1.23 1721 1.22

Consistent with the responses measured at 48 hours, the responses measured at 24 hours showed that the hPDE5 mutant construct stabilized GFP expression as evidenced by the stabilization ratios greater than one, while the wildtype construct, OT-hPDE5C-036 showed stabilization ratios around 1, indicating no ligand dependent stabilization. Results were consistent between Sildenafil and Vardenafil treatment. It was noted that the stabilization ratios obtained at 48 hours was much greater than the values obtained at 24 hours suggesting, a time dependent stabilization of GFP expression. Similar results were obtained by western blot analysis using a GFP antibody. At 0.1 μM sildenafil, modest stabilization of GFP protein levels was observed via western blot. The stabilization of GFP increased when the cells were treated with 1 μM sildenafil Both 0.1 μM and 1 μM dose of vardenafil showed stabilization of GFP levels. These trends were observed both at 24 hours and 48 hours. All comparisons were made against cells that were not treated with either ligand.

The response of hPDE5C terminal mutants to lower doses of Vardenafil was also tested and is shown in Table 38B.

TABLE 38B Dose response of hPDE5 C-terminal mutants with Vardenafil treatment Vardenafil Stabilization (nM) MFI ratio 0 137 — 1 163 1.19 3 230 1.68 10 477 3.48 30 909 6.64 100 1417 10.34 1000 1789 13.06

As shown in Table 38B, the hPDE5C terminal mutant showed a dose dependent stabilization even at lower doses of Vardenafil as indicated by the increase in the stabilization ratio. Thus, even low nano molar doses of Vardenafil are sufficient to induce ligand dependent stabilization of hPDE5 DD.

Example 20. Behavior hPDE5 Luciferase Constructs

PDE5 derived DDs were appended to reporter payload such as luciferase to generate constructs OT-hPDE5-031, OT-hPDE5-032, OT-hPDE5-033, and OT-hPDE5-035 constructs. Such constructs may be useful to study in the dynamics of destabilization and stabilization of DDs in vivo. These constructs were transduced into HCT116 cells. Cells were seeded into 96 well plates at 2000 cells per well and incubated with 1 μM or DMSO Vardenafil for 48 hours. The luciferase activity was then measured using a plate reader assay. Parental untransduced cells were used as a control to measure the background fluorescence levels. The results are shown in Table 39. In Table 39, the stabilization ratio was calculated as the fold change in luciferase signal in Vardenafil treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilizing mutation coefficient was calculated as the fold change in GFP intensity in the hPDE5 mutant constructs compared to the hPDE5 wildtype construct (OT-hPDE5-031) in the absence of the ligand. Destabilizing mutation coefficients less than 1 are desirable. Stabilization ratios greater than 1 are desired in DDs.

TABLE 39 Stability of Luciferase constructs Destabilizing Control mutation co- Stabilization Construct (DMSO) Vardenafil efficient ratio Parental cells 1367.47 3824.04 — — OT-hPDE5-031 85429.26 127499.78 — 1.49 OT-hPDE5-032 88881.46 174814.64 1.04 1.97 OT-hPDE5-033 53239.88 88918.04 0.62 1.67 OT-hPDE5-035 96460.45 269718.66 1.13 2.80

The luciferase signal intensity observed in parental cells that did not express the construct indicated that some of the signal was likely background noise. Construct, OT-hPDE5-033 showed the lowest destabilizing mutation coefficient indicating the construct is destabilized. Increase in stabilization ratios was observed with all constructs including the wildtype hPDE5 construct, OT-hPDE5-031. Construct OT-hPDE5-035 showed the highest stabilization ratio indicating the strongest ligand dependent stabilization. Among the constructs tested, OT-hPDE5-033 showed destabilization in the absence of ligand and stabilization in the presence of ligand.

Example 21. Mutagenesis of the Amino Acid 732 of hPDE5

The analysis of mutants identified by site directed mutagenesis identified amino acid hotspots whose mutation confers destabilization and ligand dependent stabilization properties to hPDE5. The amino acid at position 735 was chosen for the further analysis since the hPDE5 (R732L) mutation possessed the properties described above. To improve the DD characteristics of this DD, the amino acid at the position 732 was mutated to any of the known amino acids, including, but not limited to, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan, alanine, isoleucine, leucine, phenylalanine, valine, proline, and glycine. The library of mutations was generated by site directed mutagenesis and each of the mutants in the library was fused to a reporter protein e.g. AcGFP via a linker and transduced into HCT116 cells. The properties of the DDs were analyzed in the presence and absence of ligands via FACS as previously described. Ligands that were evaluated included, but not limited Sildenafil, and Vardenafil. The results are shown in Table 40A and Table 40B.

TABLE 40A Ligand responsive behavior of hotspot constructs Vardenafil Sildenafil 0 μM 0 μM Construct Mutation (DMSO) 1 μM (DMSO) 10 μM OT-hPDE5-001 — 161 294 989 219 OT-hPDE5-009 R732L 182 173 143 122 OT-hPDE5-064 R732G 176 854 298 288 OT-hPDE5-065 R732A 175 853 288 348 OT-hPDE5-066 R732V 150 507 189 113 OT-hPDE5-067 R732I 151 587 201 170 OT-hPDE5-068 R732P 355 310 379 361 OT-hPDE5-069 R732F 189 943 233 351 OT-hPDE5-070 R732W 301 179 248 158 OT-hPDE5-071 R732Y 302 345 397 648 OT-hPDE5-072 R732H 308 328 352 710 OT-hPDE5-073 R732S 566 399 760 746 OT-hPDE5-074 R732T 235 270 276 546 OT-hPDE5-075 R732D 294 422 298 394 OT-hPDE5-076 R732E 276 212 295 316 OT-hPDE5-077 R732Q 305 289 353 553 OT-hPDE5-078 R732N 313 290 315 516 OT-hPDE5-079 R732M 316 337 285 503 OT-hPDE5-080 R732C 526 349 499 707 OT-hPDE5-081 R732K 113 423 165 765

TABLE 40B Stabilization ratio and Destabilizing mutation coefficient of hotspot constructs Vardenafil Sildenafil Destabi- Destabi- lizing Stabili- lizing Stabili- mutation zation mutation zation Construct Mutation coefficient ratio coefficient ratio OT-hPDE5-001 — — 1.83 — 0.22 OT-hPDE5-009 R732L 1.13 0.95 0.14 0.85 OT-hPDE5-064 R732G 1.09 4.85 0.3 0.97 OT-hPDE5-065 R732A 1.09 4.87 0.29 1.21 OT-hPDE5-066 R732V 0.93 3.38 0.19 0.6 OT-hPDE5-067 R732I 0.94 3.89 0.2 0.85 OT-hPDE5-068 R732P 2.2 0.87 0.38 0.95 OT-hPDE5-069 R732F 1.17 4.99 0.24 1.51 OT-hPDE5-070 R732W 1.87 0.59 0.25 0.64 OT-hPDE5-071 R732Y 1.88 1.14 0.4 1.63 OT-hPDE5-072 R732H 1.91 1.06 0.36 2.02 OT-hPDE5-073 R732S 3.52 0.7 0.77 0.98 OT-hPDE5-074 R732T 1.46 1.15 0.28 1.98 OT-hPDE5-075 R732D 1.83 1.44 0.3 1.32 OT-hPDE5-076 R732E 1.71 0.77 0.3 1.07 OT-hPDE5-077 R732Q 1.89 0.95 0.36 1.57 OT-hPDE5-078 R732N 1.94 0.93 0.32 1.64 OT-hPDE5-079 R732M 1.96 1.07 0.29 1.76 OT-hPDE5-080 R732C 3.27 0.66 0.5 1.42 OT-hPDE5-081 R732K 0.7 3.74 0.17 4.64

The destabilizing mutation coefficients and stabilization ratios obtained for each construct with the indicated ligands were analyzed to identify constructs which possessed the lowest destabilizing mutation coefficient ratio and a second sort of the data was then performed to identify constructs with the highest stabilization ratio. This analysis allowed the identification of constructs with both the lowest destabilizing mutation coefficient that also possessed the highest stabilization ratios. Based on this analysis, it was identified that virtually all mutants tested showed destabilizing mutation coefficients less than one for the DMSO control of Sildenafil treatment, indicating that all constructs are destabilized in the absence of sildenafil. In the presence of Sildenafil, OT-hPDE5-081 construct with arginine to lysine substitution showed high sildenafil dependent stabilization ratio. Similar analysis of the constructs with Vardenafil identified OT-hPDE5-081, OT-hPDE5-066, OT-hPDE5-067, and OT-hPDE5-065 as having a low destabilizing mutation coefficient and high-ligand dependent stabilization ratios. Other constructs with low destabilizing mutation coefficients and high stabilization ratios include OT-hPDE5-067, OT-hPDE5-065, OT-hPDE5-069, OT-hPDE5-074, and OT-hPDE5-075.

The response of select mutants to increasing doses of Sildenafil, Vardenafil and Tadalafil was also tested in HCT 116 cells treated with the ligand for 48 hours. The WI values obtained with each ligand treatment are shown in Table 41A, Table 41B and Table 41C.

TABLE 41A Vardenafil Dose Response OT- OT- OT- OT- OT- Vardenafil hPDE5- hPDE5- hPDE5- hPDE5- hPDE5- (μM) 065 067 069 074 075 0 2551 1810 2188 2854 4336 0.01 2551 1810 2188 2854 4336 0.04 8345 3356 8468 6862 7350 0.12 10946 4874 10941 13187 15637 0.37 13850 7855 12987 28895 25047 1.11 15202 11777 13754 33075 37828 3.33 16783 14531 14705 41788 44499 10 18945 17645 17021 48946 50822 30 22548 24322 20693 67364 70645 1000 22548 24322 20693 67364 70645

TABLE 41B Sildenafil Dose Response OT- OT- OT- OT- OT- Sildenafil hPDE5- hPDE5- hPDE5- hPDE5- hPDE5- (μM) 065 067 069 074 075 0.01 808 579 688 856 1192 0.04 1025 612 916 1041 1270 0.12 1270 592 1205 1113 1337 0.37 1931 771 2002 1835 1867 1.11 2655 1040 2711 3355 3108 3.33 3769 1709 3555 6896 6171 10 4716 2733 4427 12407 13971 30 5694 4197 5449 21258 21120 1000 5694 4197 5449 21258 21120

TABLE 41C Tadalafil Dose Response OT- OT- OT- OT- OT- Tadalafil hPDE5- hPDE5- hPDE5- hPDE5- hPDE5- (μM) 065 067 069 074 075 0.01 752 498 631 825 1163 0.14 1259 540 1193 1068 1361 0.41 2066 712 2075 1655 1548 1.23 3412 1041 3134 3433 2504 3.7 4690 1745 4294 6697 4821 11.11 5126 2746 4734 12843 10583 33.33 6264 3970 5293 17164 16956 100 6301 5487 5867 21113 22630 1000 6301 5487 5867 21113 22630

OT-hPDE5-074 with an arginine to threonine substitution showed the strongest increase in GFP expression at the highest dose, with all three ligands tested. At doses lower than 1 μM of Vardenafil, all constructs showed ligand dependent stabilization. At doses of Sildenafil lower than 1 μM, OT-hPDE5-069, OT-hPDE5-074 and OT-hPDE5-075 showed ligand dependent stabilization. With doses of Tadalafil lower than 1 OT-hPDE5-065, OT-hPDE5-069 and OT-hPDE5-074 showed ligand dependent stabilization. By mutagenesis of the R732 locus, DDs with a variety of properties were obtained. The selection of a particular DD may be made based on the payload utilized and the extent of expression desired with the payload. The EC50 values for each of the constructs with each of the ligands is shown in Table 41D.

TABLE 41D EC₅₀ for Hotspot Mutants OT- OT- OT- OT- OT- hPDE5- hPDE5- hPDE5- hPDE5- hPDE5- Ligand 065 067 069 074 075 Vardenafil 0.30 2.20 0.28 1.47 1.44 Sildenafil 2.08 6.90 1.76 7.50 7.04 Tadalafil 1.45 14.28 1.33 8.64 14.54

These results are consistent with the western blot analysis of GFP protein levels in HCT 116 cells stably transduced with these mutants. The following constructs showed a strong stabilization of GFP levels in the presence of vardenafil and when compared to DMSO controls: OT-hPDE5-009, OT-hPDE5-064, OT-hPDE5-065, OT-hPDE5-068, OT-hPDE5-070, OT-hPDE5-071, OT-hPDE5-072, OT-hPDE5-073, OT-hPDE5-074, OT-hPDE5-075, OT-hPDE5-076, OT-hPDE5-077, OT-hPDE5-078, OT-hPDE5-079, and OT-hPDE5-080. Modest ligand dependent stabilization of GFP levels was observed with OT-hPDE5-066, OT-hPDE5-067, OT-hPDE5-069, and OT-hPDE5-081. All samples showed equal protein loading as measured by GAPDH protein levels.

Example 22: Dynamic Range of hPDE5 Regulation

HCT116 cells were transduced with hPDE5-GFP constructs, OT-hPDE5-083, OT-hPDE5-084, OT-hPDE5-085, and OT-hPDE5-094 and incubated with the indicated concentrations of ligand for 48 hours. GFP fluorescence was measured by FACS. The fold change in GFP expression over untreated DMSO control is shown in Table 42A, Table 42B and Table 42C for vardenafil, sildenafil and tadalafil respectively.

TABLE 42A Vardenafil dose response Vardenafil OT-hPDE5- OT-hPDE5- OT-hPDE5- OT-hPDE5- (log) 83 84 85 94 1.477 20.664 13.612 4.507 16.171 1.000 18.207 12.030 2.178 13.462 0.523 18.327 10.260 1.277 13.230 0.046 17.229 7.465 1.078 12.550 −0.431 16.143 4.374 1.022 11.992 −0.908 13.764 2.258 1.025 9.770 −1.386 10.108 1.323 1.015 5.477 −1.863 4.673 0.834 1.030 2.303 −2.340 2.575 0.741 1.034 1.412 −2.817 1.744 0.725 1.047 1.105

TABLE 42B Sildenafil dose response Sildenafil OT-hPDE5- OT-hPDE5- OT-hPDE5- OT-hPDE5- (log) 83 84 85 94 2.000 11.370 14.212 1.909 11.290 1.523 12.925 15.247 1.509 13.091 1.046 12.066 10.028 1.212 12.344 0.569 10.587 5.408 1.091 10.615 0.092 9.190 2.822 0.995 8.165 −0.386 6.849 1.640 1.040 5.536 −0.863 4.225 1.249 0.993 2.540 −1.340 2.295 1.106 1.007 1.397 −1.817 1.434 1.025 1.013 1.134 −2.294 1.136 1.024 0.966 0.997

TABLE 42C Tadalafil dose response Tadalafil OT-hPDE5- OT-hPDE5- OT-hPDE5- OT-hPDE5- (log) 83 84 85 94 2.000 12.074 14.919 1.498 12.606 1.523 11.481 13.740 1.224 11.910 1.046 9.897 8.083 1.011 11.144 0.569 8.403 4.360 0.945 9.902 0.092 5.445 2.312 0.973 7.992 −0.386 2.725 1.472 0.907 5.049 −0.863 1.563 1.225 0.946 2.887 −1.340 1.121 1.034 0.938 1.402 −1.817 1.095 1.013 0.976 1.077 −2.294 1.045 0.985 0.942 1.064

As shown in Table 42A, Table 42B and Table 42C, dynamic regulation of hPDE5 DDs was observed with OT-hPDE5-083, OT-hPDE5-084, and OT-hPDE5-085. The hPDE5 wildtype construct shown little to no ligand dependent stabilization with all three ligands. The stabilization concentration 50 or (SC₅₀), which is the concentration of ligand required to achieve 50% stabilization, was also calculated for all four constructs with each of the ligands and the results are shown Table 42D.

TABLE 42D Stabilization Concentration 50 (SC₅₀) of hPDE5 DDs OT-hPDE5- OT-hPDE5- OT-hPDE5- OT-hPDE5- Ligand 83 84 85 94 Vardenafil 0.04 1.15 >30 0.07 Sildenafil 0.38 6.82 >30 0.67 Tadalafil 1.97 9.54 >100 0.77

Taken together these results indicate that structure-guided mutagenesis of ligand-binding site in PDE5 generates 23 nM ligand-DD interaction. OT-hPDE5-083 with Y612F, R732L, was able to bind to Vardenafil with SC₅₀, of 23 nM suggesting potent DD-ligand interaction.

Example 23. DD Regulated IL15-IL15-Ra Expression In Vitro

Immunotherapeutic payloads of the invention such as, IL15-IL15Ra fusion proteins, were fused to any of the DDs described herein and cloned into expression vectors, such as pLVX and pELNS vectors. To test ligand dependent IL15-IL15Ra production, HEK293T cells were transiently transfected with the constructs. Cells were then plated in DMEM with 10% FBS and incubated overnight at 37° C., 5% CO2. The growth media was exchanged for media containing ligand, e.g. Vardenafil at 10 μM. Following 48-hour incubation with ligand, cells were lysed and immunoblotted using antibodies specific to IL15Ra. hPDE5-DDs were able to regulate IL15Ra levels in the presence of Vardenafil with all three constructs tested OT-IL15-043, OT-IL15-044 and OT-IL15-045. In contrast, the IL15Ra levels remained unchanged both in the presence and absence of ligand with the constitutive construct, OT-IL15-008. It was noted that the ligand dependent stabilization was accompanied by an upward shift in the mobility of IL15Ra only in the presence of Vardenafil, indicating that the stabilization is likely accompanied by a post translation protein modification. Uniform loading of samples was demonstrated using actin as a loading control.

The effects of vector backbone on the expression of the payload were examined by cloning the OT-IL15-031 construct into the pELPS vector backbone to generate the OT-IL15-079 construct. The constructs were transduced into HEK293T cells and IL15Ra expression was assayed as described above. Only OT-IL15-031 showed vardenafil dependent stabilization of IL15Ra protein levels. Only modest ligand dependent stabilization was observed with the OT-IL15-079 construct in the presence of vardenafil.

The IL15 levels were also examined using the MSD assay. The growth media was harvested from the cells expressing IL15-IL15Ra and that were exposed to 10 μM Vardenafil or vehicle control for 48 hours. The results are shown in Table 43, where the stabilization ratio was defined as the ratio of expression, function or level of a protein of interest, i.e. IL15-IL15Ra in response to the stimulus, i.e. Vardenafil; to the expression, of the IL15-IL15Ra in the absence of the stimulus, i.e. DMSO control. The destabilization ratio was calculated as the ratio of expression, of IL15Ra in the absence of the stimulus specific to the effector module i.e. Vardenafil to the expression, function or level of the protein of interest, that is expressed constitutively (OT-IL15-008) and in the absence of the stimulus specific to the SRE. Stabilization ratios greater than one and destabilization ratios less than one are desired.

TABLE 43 IL15 levels (pg/ml) Destabilization Stabilization Construct Control Vardenafil ratio ratio OT-IL15-008 710.73 799.39 — 1.12 OT-IL15-043 63.37 93.63 0.09 1.48 OT-IL15-044 193.09 203.21 0.27 1.05 OT-IL15-045 255.05 320.39 0.36 1.26

As shown in Table 43, all three hPDE5 regulated constructs showed destabilization ratios less than one indicating destabilization in the absence of ligand. OT-IL15-043 appeared to be the most destabilized construct. It was also the most ligand-stabilized construct, with the highest stabilization ratio.

Expression of OT-IL15-031 construct was also analyzed in HCT-116 cells via western blot. The cells were exposed to 10 μM Vardenafil for 48 hours and IL15 and IL15Ra levels were analyzed by western blot. Similar to the HEK293T cells, HCT 116 cells also displayed a Vardenafil dependent stabilization with a concomitant upward shift in mobility of IL15Ra protein indicating protein modifications.

Example 24. Effect of Linker on DD Regulation of IL15-IL15Ra

The effect of the type of linker used and the length of the linker on the regulation of the expression of the payload was tested. IL15-IL15Ra fusion constructs were linked to the hPDE5 (R732L) DD using GSGSGS (SEQ ID NO. 8330) as the linker (as in OT-IL15-111), or GSGSGSGS (SEQ ID NO. 8331) as the linker (as in OT-IL15-112) or using GSGSGGGSGS (SEQ ID NO. 8332) as the linker (as in OT-IL15-113). The IL15 portion of the construct was tagged with a Flag tag while the IL15Ra portion was tagged with a HA tag. These tags allow both the components of the construct to be tracked individually in experiments. The constructs were transiently transfected into HEK293T cells and then incubated with 1 μM Vardenafil for 24 hours. The properties of the DDs regulated IL15-IL15Ra payloads were analyzed in the presence and absence of ligands via FACS using the HA antibody as previously described. The results are shown in Table 44, where the stabilization ratio was defined as the ratio of expression, function or level of a protein of interest, i.e. IL15-IL15Ra in response to the stimulus, i.e. Vardenafil; to the expression, of the IL15-IL15Ra in the absence of the stimulus, i.e. DMSO control treated samples.

TABLE 44 % HA positive cells with different linkers Stabilization Construct Name Control Vardenafil ratio OT-IL15-111 37.5 63.2 1.69 OT-IL15-112 41.6 74.6 1.79 OT-IL15-113 42 69.1 1.65

As shown in Table 44, all three constructs each with a different linker resulted in a somewhat similar stabilization ratio, indicating that linker length and identity did not impact regulation of the payload by the DD. All three constructs also had a stabilization ratio greater than one suggesting that the payload expression was stabilized in the presence of the ligand. Among the three constructs tested OT-IL15-112 showed a slightly higher stabilization ratio than the other two constructs indicating that the use of the GSGSGSGS linker may result in a slightly improved ligand dependent stabilization.

Example 25. DD Regulated CD19 CAR Expression and Function

Immunotherapeutic payloads of the invention such as CD19 CAR were fused to any of the DDs described herein and cloned into expression vectors pLVX and pELNS vectors. In this manner, OT-CD19-052 (hPDE5 (WT)) and OT-CD19-053 (hPDE5 (R732L)). The HA tag was added to enable the easy detection of the chimeric proteins.

To test ligand dependent CD19 CAR production, NIH 3T3 cells were transiently transfected with the constructs. Cells were then plated in a growth media e.g. DMEM with 10% FBS and incubated overnight at 37° C., 5% CO2. The growth media was exchanged for media containing ligand, at various concentrations of Vardenafil ranging from 30 nM to 10,000 nM. Following 48-hour incubation with ligand, cells were analyzed by FACS using the HA antibody and the mean fluorescence intensity (MFI) was calculated. The results are shown in Table 45A, where the stabilization ratio was defined as the ratio of expression, function or level of a protein of interest, i.e. CD19 CAR in response to the stimulus, i.e. Vardenafil; to the expression, of the CD19 CAR in the absence of the stimulus, i.e. DMSO control. Stabilization ratios greater than one are desired.

TABLE 45A CD19 CAR expression OT-CD19- OT-CD19-053 (R732L) Vardenafil 052 (WT) Stabilization (nM) MFI MFI ratio 0 0 1336 — 30 9125 3066 2.29 100 9592 4674 3.50 300 10509 5732 4.29 1000 10328 6738 5.04 10000 9285 3931 2.94

As shown in Table 45A, a stabilization ration greater than 1 was observed with all doses of Vardenafil, and even with the lowest concentration of Vardenafil (30 nM) suggesting that low doses of ligand may be sufficient to achieve ligand dependent stabilization. It was also noted that the stabilization ratio obtained at the highest concentration of ligand i.e. 10,000 nM was lower than the ratio obtained with 1000 nM ligand suggesting a bimodal pattern of stabilization.

The dose dependent regulation of OT-CD19-052 and OT-CD19-053 was measured in HEK 293 cells transfected with 2 ug DNA and stably selected using 2 ug/ml puromycin. CAR surface expression was detected using Protein L staining following 24 hours of ligand treatment at the indicated concentrations. The median Protein L fluorescence is shown in Table 45B.

TABLE 45B Ligand dose dependent CAR expression Tadalafil Sildenafil Vardenafil OT- OT- Empty OT- OT- Empty OT- OT- Empty Ligand CD19- CD19- Vector CD19- CD19- Vector CD19- CD19- Vector (nM) 052 053 (pLVX) 052 053 (pLVX) 052 053 (pLVX) 0.15 21122 4064 77 14703 2848 89.8 17946 4202 87.9 0.46 15335 3501 75.8 15335 3304 87 17746 4064 92 1.39 14662 3501 78.8 13143 3250 83.9 16175 4604 89.8 4.22 14377 3341 77 13901 2810 85.7 16450 5977 93.9 12.79 10773 3599 78.8 9419 2976 81.8 17646 9210 92 38.74 14098 4042 80 12083 3268 84.8 16175 13329 94.8 117.41 18354 6302 77 15037 3589 83 19856 20594 93.9 355.78 13785 11235 72.9 13901 5274 83.9 22406 23175 95.8 1078.11 18200 15640 80 13708 8054 88.8 22343 31138 97.7 3267.00 15950 21301 75.8 14337 13291 84.8 24933 31226 100 9900.00 21421 24654 75.8 17746 23371 84.8 26824 36249 110 30000.00 27902 33691 77 25003 25215 84.8 30617 35945 140

As shown in Table 45B, only OT-CD19-053 responded to increasing doses of ligand with increase surface CAR expression, but not the WT construct OT-CD19-052. This was observed with all three ligands. However, the highest levels of CAR expression were observed with increasing doses of vardenafil. The EC50 for vardenafil, tadalafil and sildenafil were determined to 25 nM, 380 nM, and 1500 nM respectively.

Cells were also treated with ligand for varying durations of time with ligand concentrations shown in Table 45C and Protein L expression was measured using FACS. The results are shown in Table 45C and Table 45D. Stabilization ratios are shown in Table 45E.

TABLE 45C Time dependent increase in CAR expression with Vardenafil Vardenafil Vardenafil 10 μM 1 uM DMSO Empty OT- OT- OT- OT- OT- Empty Vector CD19- CD19- CD19- CD19- CD19- Vector Hours (pLVX) 052 053 053 052 053 (pLVX) 0 1412 5188 1486 65.9 5060 1386 71.9 2 2863 11643 2918 78.7 5545 1270 71 4 3654 11258 3526 76.8 5408 1274 71.9 24 6624 10439 8005 83.7 5845 1241 71 48 7764 8118 8489 93.8 6128 1287 71 72 9282 7961 9788 92.8 6442 1329 65.9

TABLE 45D Time dependent increase in CAR expression with Tadalafil and Sildenafil Tadalafil Tadalafil Sildenafil Sildenafil 10 μM 1 μM 10 μM 1 μM Empty OT- OT- OT- Empty OT- OT- OT- Vector CD19- CD19- CD19- Vector CD19- CD19- CD19- Hours (pLVX) 052 053 053 (pLVX) 052 053 053 0 65 4909 1336 1368 62.8 5469 1534 1372 2 65 5911 2778 2451 65 8536 2801 2278 4 65 6196 3032 2950 63.7 8560 3256 2617 24 65 8608 6606 4346 62.8 8118 5499 3159 48 62.8 8827 7384 4683 62.8 7404 5927 3283 72 58.7 8118 8050 6027 58.7 6111 5188 3506

TABLE 45E Time dependent increase in CAR expression Tadalafil Tadalafil Sildenafil Sildenafil Vardenafil Vardenafil 10 μM 1 μM 10 μM 1 μM 10 μM 1 μM OT- OT- OT- OT- OT- OT- OT- OT- OT- Time CD19- CD19- CD19- CD19- CD19- CD19- CD19- CD19- CD19- (hrs) 052 053 053 052 053 053 052 053 053 0 0.97 0.96 0.99 1.08 1.11 0.99 1.03 1.07 0.05 2 1.07 2.19 1.93 1.54 2.21 1.79 2.10 2.30 0.06 4 1.15 2.38 2.32 1.58 2.56 2.05 2.08 2.77 0.06 24 1.47 5.32 3.50 1.39 4.43 2.55 1.79 6.45 0.07 48 1.44 5.74 3.64 1.21 4.61 2.55 1.32 6.60 0.07 72 1.26 6.06 4.53 0.95 3.90 2.64 1.24 7.36 0.07

The analysis of the stabilization ratios indicated that OT-CD19-053 is stabilized by all three ligands with increased duration of incubation time with ligand. As expected, 10 μm dose of ligand showed much greater expression of CAR than the 1 μM dose. Regulation was evident even at the lower doses.

Constructs OT-CD19-130, OT-CD19-131 and OT-CD19-132 were cloned into pELNS vectors and transduced into T cells. Three different volumes of lentiviral supernatant were tested on cells i.e. 1 μl, 10 μl and 20 μl. Following transduction, cells were treated with 10 μM Vardenafil or left untreated for 24 hours beginning on day 4. Untransduced cells were also included as negative control. On day 5, the percentage CAR positive cells were measured by FACS using 1 μg/ml CD19 Fc. The results are shown in FIG. 19A.

As shown in FIG. 19A, all three constructs showed a viral dose dependent increase in the percentage of CAR positive cells, in the presence of Vardenafil. Although similar trends were observed in untreated controls, the percentage of CAR positive cells observed with no vardenafil treatment was substantially lesser than the vardenafil treated controls. Among the three constructs, OT-CD19-132 showed lowest basal expression of the CAR in the absence of ligand and the highest percentage of CAR positive cells in the presence of vardenafil. Similar observations were made when comparing the percentage CAR positive cells obtained with different constructs using 20 μl of virus as shown in FIG. 19B, where a shift in the number of cells that are CAR positive was observed in vardenafil treated cells (labelled treatment) compared to untransduced as well as untreated cells.

T cells maintained in culture for over 10 days reach quiescence and accordingly T cells transduced with OT-CD19-131 did not show vardenafil dependent CAR expression at day 11. However, restimulation of the same T cells on day 11 with CD3/CD28 beads (at 3:1 bead: cell ratio, for 24 hours concomitant with ligand treatment) restored vardenafil dependent CAR expression in T cells transduced with 20 μl of virus (FIG. 19C). The effect of restimulation on the expression of the CAR was evident at all concentrations of the virus tested FIG. 19D. Taken together, these data show that CD19 CARs operably linked to hPDE5 DDs demonstrate ligand dependent CAR expression. Further, ligand responsiveness and subsequent CAR expression may be restored in quiescent T cells by restimulation with CD3/CD28 beads.

To test the effect on different ligands on hPDE5-CAR constructs, the constructs were packaged into plasmids and transduced into T cells using 10 μl of virus. On day 9, T cells expressing either OT-CD19-111, OT-CD19-130, OT-CD19-131 or OT-CD19-132 were treated with varying doses of ligand ranging from 0.1 nM to 10 μM for 24 hours. The constitutive construct OT-CD19-063 was included as the positive control, and the cells transduced with the empty vector (pELNS) served as the negative control. Three different ligands for hPDE5 were tested including, Sildenafil, Tadalafil and Vardenafil. Cells were analyzed by FACS and sorted for singlets/live CAR positive cells. The results are shown in FIG. 19E. Among the three ligands tested, all constructs were most responsive to Vardenafil, followed by Tadalafil and Sildenafil. As seen previously, the OT-CD19-131 and OT-CD19-132 showed lower basal expression, and had the higher EC50 values than the other constructs. The experiments were repeated with the OT-CD19-131 construct by transducing T cells with different volumes of virus transduced into T cells using 10 μL, 2 μL, 0.4 μL, or 0.084, of virus and the CAR expression was measured by FACS, both in the presence of all three ligands and in the absence of the ligands. The results are shown in Table 45F, where “basal % CAR+” indicates the percentage of CAR positive cells in the absence of ligand, and the “Max % CAR+,” indicates the maximum expression of the CAR in the presence of ligand. The EC50 is defined as the concentration of a drug/ligand that gives half-maximal response. The response herein refers to the expression of the chimeric antigen receptor.

TABLE 45F hPDE5 regulation of CD19-CAR Virus Varden- Tadal- Silden- Vol- Basal Max afil afil afil ume % % EC50 EC50 EC50 Construct (uL) CAR+ CAR+ (nM) (nM) (nM) Empty vector 1 0.2 0.2 — — — (pELNS) OT-CD19-063 1 30.0 30.0 — — — OT-CD19-131 10 3.6 37.5 140.0 1312.2 >10000.0 OT-CD19-131 2 2.0 29 157.0 1402.8 >10000.0 OT-CD19-131 0.4 0.9 16.1 197.7 2511.9 >10000.0 OT-CD19-131 0.08 0.4 5.0 220.8 1857.8 >10000.0

Among the three ligands tested, higher transduction volume increased the maximum CAR expression level achieved, but did not alter ligand EC50 values.

To test how rapidly hPDE5-CAR can be stabilized with ligand, the indicated constructs were tested in T cells. T cells from donor were thawed and activated overnight in the presence of CD3/CD28 Dynabeads at a 3:1 bead: cell ratio. The following day, cells were transduced with 104, lentivirus prepared from OT-CD19-063, OT-CD19-111, OT-CD19-130, OT-CD19-131, and OT-CD19-132 constructs. Cells were expanded by addition of fresh media over the course of 10 days, maintaining cells around 0.5×10{circumflex over ( )}6 cells/mL, then frozen. Expanded T cells were thawed and restimulated with CD3/CD28 beads for 48 hours. Cells were treated with 10 μM Tadalafil or Vardenafil at various times such that all conditions received 48 hr of bead stimulation with either 0, 2, 4, 6, 24, or 48 hours of ligand treatment. CAR surface expression was measured with 1 μg/mL CD19-Fc. The results are shown in FIG. 19F. In activated T cells, CAR surface expression occurred at nearly maximum levels by 2 hours after ligand addition.

To test whether CD19-CARs showing regulated expression also show regulated cytotoxicity, transduced T cells were tested in a cytotoxicity assay against the CD19-expressing Nalm6 tumor cell line, T cells from a human donor were thawed and activated overnight in the presence of CD3/CD28 Dynabeads at a 3:1 bead: cell ratio. The following day, cells were transduced with 10 μL lentivirus from OT-CD19-063, OT-CD19-111, OT-CD19-130, OT-CD19-131, and OT-CD19-132. Cells were expanded by addition of fresh media over the course of 10 days, maintaining cells around 0.5×10{circumflex over ( )}6 cells/mL, then frozen. Expanded T cells were thawed and cocultured with CD19 expressing target cells, Nalm6-Katushka, at effector: target cell ratio of 10:1 for 6 days, in the absence or presence of Tadalafil. Images captured on the Incucyte Zoom were analyzed for proliferation of target cells by measuring red fluorescence over time (Total NucRed Area). In the absence of ligand, i.e. DMSO, OT-CD19-131 and OT-CD19-132 showed target cell proliferation as measured by the increase in total NucRed area (red fluorescence). Constructs, OT-CD19-130, OT-CD19-111 showed very low levels of red fluorescence indicating basal activity that was comparable to the constitutively expressed CAR construct OT-CD19-063. All constructs tested showed a reduction in red fluorescence when treated with either 3 μM Tadalafil or 10 μM Tadalafil that was comparable to the red fluorescence levels observed with the constitutively expressed CAR. As expected, the untransduced cells showed high levels of red fluorescence both in the presence and absence of ligand.

Constructs showing higher basal CAR expression in T cells, OT-CD19-111 and OT-CD19-130, inhibited target cell growth in the absence of tadalafil. T cells transduced with OT-CD19-132, which had the highest Tadalafil EC50 in terms of CAR expression, required higher concentrations of ligand to generate an in vitro functional response.

To measure cytokine levels in T cells, supernatants from the previous coculture assay were harvested at 48 hours and analyzed for IFNγ and IL2 levels by MSD assay (Meso Scale Discovery's ELISA). The results are shown in FIG. 19G. The cytokine levels at the following effector to target cell rations: 10:1, 3:1, 1:1, 0.3:1, and 0.1:1. High levels of both IFNγ and IL2 are produced by OT-CD19-131 transduced T cells when co-cultured with Nalm6 targets only in the presence of 3 μM Tadalafil, correlating with the cytotoxicity observed.

To confirm the cytotoxicity and cytokine secretion is directly related to CAR expression, OT-CD19-131 transduced T cells were cocultured with Nalm6-Katushka target cells at 10:1 effector: target cell ratio for 6 days in the presence of a dose titration of Tadalafil from 10 μM to 10 nM. The fluorescence of Nalm6 cells was measured over a period of 6 days in response to the following doses of tadalafil: 0.01 μM, 0.04 μM, 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, 10 μM. Tadalafil doses at 0.37 μM, 1.11 μM, 3.33 μM, 10 μM showed a reduction in Nalm6 proliferation over 6 days. Lower concentrations (<0.37 μM) tadalafil did not cause a reduction in Nalm6 fluorescence, in fact, Nalm6 cells continue to proliferate over the span of 6 days. Significant CAR expression was detected by FACS using CD19-Fc at Tadalafil concentrations at or above 300 nM. This CAR expression corresponded to Tadalafil dose-dependent cytotoxicity (FIG. 19H) and cytokine secretion of IFNγ and IL2 (FIG. 19I).

The functionality of the CAR transduced T cells was also tested in vitro. T cells were transduced with OT-CD19-131 or the constitutive construct OT-CD19-063. Cells were then frozen. Prior to the experiment, T cells were thawed overnight, and cocultured with Nalm6 target cells stably expressing Katushka RFP. Nalm6 cells express high levels of CD19 antigen and are hence ideal target cells in cell killing assay that test CD19 CAR constructs. T cells were mixed with Nalm6 cells at an effector cell to target cell (E: T) ratio of 5:1 for 12 hours in the presence of 10 μM vardenafil or DMSO. Target cell apoptosis was determined by measuring Annexin V fluorescence over time using the Incucyte instrument. Cell killing is measured as the ratio of the total killed target area and to the NALM6-Katushka area (μM²). As shown in FIG. 19J, the total killed target cell area increased when Nalm6 cells were co-cultured with OT-CD19-131 expressing T cells in the presence of vardenafil. Similar trend was observed in the proliferation of Nalm6 cells (FIG. 19K). The same cells did not result in significant cell killing in presence of DMSO, showing that the target cell killing by the T cells is specifically in response to the presence of the ligand. As expected, the constitutive construct, OT-CD19-063 showed increased total killed target cell area both in the presence and absence of ligand, with a trend towards more killing in the presence of the ligand. Nalm6 cells co-cultured with untransduced T cells and Nalm6 cells that were not co-cultured with T cells did not show an increase in the total killed target area. Taken together, these data show that the regulated CAR construct, OT-CD19-131 is active on antigen positive cells only in response to ligand.

The cell supernatants were also collected from the T cell/Nalm6 cytotoxicity co-culture assays at 66 hours and Interferon gamma levels were measured by MSD assays. The results are shown in Table 45G.

TABLE 45G Interferon gamma production (pg/mL) Construct DMSO Vardenafil 10 μM OT-CD19-131 443.99 2282.98 OT-CD19-063 3268.72 1980.48 Untransduced 16.57 18.48

As shown in Table 45G, the interferon gamma levels showed a five-fold induction in OT-CD19-131 transduced T cells with the addition of vardenafil. Vardenafil treatment did not induce interferon gamma production in OT-CD19-063 transduced T cells or untransduced T cells, indicating that the ligand specific induction of interferon gamma is related to the increased CAR expression and cell killing observed under similar conditions. The interferon gamma levels in DMSO treated OT-CD19-131 was lower than the levels observed with the constitutive construct indicating that the OT-CD19-131 cells have low basal expression of the CD19 CAR.

Example 26. In Vivo Assessment of hPDE5-Regulated CD19 CAR

To test the efficacy of ligand regulated CAR efficacy in a tumor rejection model, 5 million CAR positive T cells (25 million total T cells) were injected in Nalm6 tumor bearing female NSG mice. The Nalm6-Luc is a B-cell precursor leukemia cell line used to generate an intravenous disseminated tumor model for studying blood tumors. Mice were treated daily with vehicle or Tadalafil at the following mg/kg body weight or mpk: 10 mpk, 30 mpk, or 100 mpk. Constitutive OT-CD19-063 transduced T cells served as a positive control and untransduced T cells as a negative control. T cells were injected 7 days after the Nalm6 cells were implanted. Animals in the Tadalafil groups received 2 doses of ligand prior to T cells being injected. The objective of the study was to test the ligand dose response regulation of a hPDE5 DD CD19 CAR-T and its impact on efficacy. Further, the study was also used to address if the presence of antigen was necessary for detection and expression of the CAR. Dose groups included (a) untransduced T cells (b) untransduced vehicle treated cells (c) Un-transduced cells dosed with Tadalafil 100 mg/kg (d) 5.0 Million CD19+OT-CD19-063 CAR T cells (e) 5.0 Million CD19 positive Nalm6 cells with OT-CD19-063 CAR T cells (Vehicle) (f) 5.0 Million CD19 positive Nalm6 cells with OT-CD19-063 CAR T cells (Tadalafil 100 mg) (g) 5.0 Million CD19 positive Nalm6 cells with OT-CD19-131 CAR T cells (Vehicle) (h) 5.0 Million CD19 positive Nalm6 cells with OT-CD19-131 (Tadalafil 10 mg/kg) (i) 5.0 Million CD19 positive Nalm6 cells with OT-CD19-131 CAR T cells (Tadalafil 30 mg/kg (j) 5.0 Million CD19 positive Nalm6 cells with OT-CD19-131 CART cells (Tadalafil 100 mg/kg). All animals were dosed once a day, orally for 25 days. Tumor burden assessments were made biweekly using bio luminescent imaging and the fluorescence intensity of Nalm6 Luc cells was observed over time. Two terminal collections were made to assess bone marrow, blood and spleen cell populations. Reduction in tumor burden/prevalence was measured by decreased luminescence from Nalm6-Luciferase cells, and was observed in Tadalafil treated CD19-131 animals (FIG. 20A). In FIG. 20A, “const” refers to OT-CD19-063 and “regulated” refers to OT-CD19-131. Taken together these data show that daily oral dosing of tadalafil resulted in dose-dependent suppression of tumor growth and/or reduction in tumor burden. Maximal tumor suppression was comparable to constitutive CAR with very low activity in the absence of drug. Plasma PK was also established in non-tumor-bearing mice of the hPDE5 inhibitors Tadalafil and Vardenafil after a single dose at the indicated dose levels and is shown in FIG. 20B and FIG. 20C. 

1-51. (canceled)
 52. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one payload which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), wherein the DD comprises the catalytic domain of a human cGMP-specific 3′,5′-cyclic phosphodiesterase (hPDE5), wherein the DD comprises SEQ ID NO: 3, and wherein the DD further comprises a mutation in the amino acid at the position corresponding to amino acid 732 (R732) of SEQ ID NO:
 1. 53. The composition of claim 52, wherein the mutation in the amino acid at position 732 (R732) is selected from the group consisting of R732L, R732A, R732G, R732V, R732I, R732P, R732F, R732W, R732Y, R732H, R732S, R732T, R732D, R732E, R732Q, R732N, R732M, R732C, and R732K.
 54. The composition of claim 53, wherein the mutation in the amino acid at position R732 is R732L.
 55. The composition of claim 52, wherein the DD further comprises a mutation in the amino acid at the position corresponding to amino acid 764 (D764) of SEQ ID NO: 1, wherein the mutation at D764 is selected from the group consisting of D764N and D764A.
 56. The composition of claim 52, wherein the DD further comprises a mutation in the amino acid at the position corresponding to amino acid 612 (Y612) of SEQ ID NO: 1, wherein the mutation at Y612 is selected from the group consisting of Y612A, Y612F, and Y612W.
 57. The composition of claim 52, wherein the DD further comprises an F736A mutation in the amino acid at the position corresponding to amino acid 736 (F736) of SEQ ID NO:
 1. 58. The composition of claim 52, wherein the DD further comprises an H653A mutation in the amino acid at the position corresponding to amino acid 653 (H653) of SEQ ID NO:
 1. 59. The composition of claim 52, wherein the payload is an immunotherapeutic agent.
 60. The composition of claim 59, wherein the immunotherapeutic agent is selected from a chimeric antigen receptor (CAR), a cytokine, a cytokine-cytokine receptor fusion polypeptide.
 61. The composition of claim 60, wherein the immunotherapeutic agent is a chimeric antigen receptor (CAR) that comprises: (a) an extracellular target moiety; (b) a transmembrane domain; (c) an intracellular signaling domain; and (d) optionally, one or more co-stimulatory domains.
 62. The composition of claim 61, wherein the extracellular target moiety is a scFv derived from an antibody that specifically recognizes a CD19 target molecule.
 63. The composition of claim 61, wherein the immunotherapeutic agent is a cytokine selected from the group consisting of IL2, IL12 and IL15.
 64. The composition of claim 61, wherein the immunotherapeutic agent is a cytokine-cytokine receptor fusion polypeptide that comprises the whole or a portion of IL15, fused to the whole or a portion of IL15Ra to produce a IL15-IL15Ra fusion polypeptide.
 65. The composition of claim 52, wherein the SRE is responsive to one or more stimuli, and wherein said one or more stimuli is selected from Tadalafil, Vardenafil, Sildenafil, Avanafil, Lodenafil, Mirodenafil, Udenafil, Benzamidenafil, Dasantafil, and Beminafil.
 66. A pharmaceutical composition comprising the composition of claim 52 and a pharmaceutically acceptable excipient.
 67. A polynucleotide encoding the composition of claim
 52. 68. A pharmaceutical composition comprising the polynucleotide of claim 67 and a pharmaceutically acceptable excipient.
 69. A vector comprising the polynucleotide of claim
 67. 70. A cell comprising the polynucleotide of claim
 67. 71. The cell of claim 70, wherein said cell is an immune cell for adoptive cell transfer (ACT), and optionally wherein the immune cell is a CD8⁺ T cell, a CD4⁺ T cell, a memory T cell, a terminally differentiated effector T cell, a natural killer (NK) cell, a NK T cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T lymphocyte (CTL), a regulatory T cell (Treg), or a dendritic cell (DC).
 72. A pharmaceutical composition comprising the cell of claim 70 and a pharmaceutically acceptable excipient.
 73. A method of producing a modified cell, said method comprising introducing into a cell a nucleic acid molecule comprising the polynucleotide of claim
 67. 74. A method of modulating expression, function, and/or level of a payload in the cell of claim 70, said method comprising: administering to the cell a stimulus, wherein the SRE is responsive to the stimulus and wherein the expression, function, and/or level of the payload is modulated in response to the stimulus.
 75. A method of inducing an immune response in the immune cell of claim 71, wherein the payload is an immunotherapeutic agent, said method comprising: administering to the cell a therapeutically effective amount of a stimulus to modulate the expression of the immunotherapeutic agent, wherein the immunotherapeutic agent is capable of inducing an immune response in the cell in response to the stimulus.
 76. A method of reducing a tumor burden in a subject, comprising: (a) administering to the subject a therapeutically effective amount of the immune cells of claim 71; and (b) administering to the subject a therapeutically effective amount of a stimulus, wherein the stimulus is a ligand, to modulate the expression of the immunotherapeutic agent, thereby reducing the tumor burden.
 77. A method of treating a disease in a subject in need thereof, said method comprising: (a) administering to the subject a therapeutically effective amount of the cell of claim 70, wherein the cell comprises a payload that treats the disease; and (b) administering to the subject a therapeutically effective amount of a stimulus, wherein the SRE is responsive to the stimulus and wherein expression of the payload is modulated in response to the stimulus to thereby treat the disease. 